Plant resistance gene

ABSTRACT

Disclosed are isolated nucleic acids consisting essentially of RPW nucleotide sequences (especially RPW8 from  Arabidopsis thaliana , and related homologues and other sequences e.g. from  Brassica napus; B. oleracea ). These encode a novel class of resistance polypeptides having an N-terminal transmembrane domain and a coiled coil domain and which is capable of recognising and activating in a plant into which said nucleic acid is introduced a specific defense response to challenge with a powdery mildew pathogen e.g.  E. cichoracearum . Also provided are related products e.g. primers, polypeptides, transgenic plants having enhanced resistance, plus also processes for producing these, and methods of use.

[0001] The present invention relates to methods and materials,particularly nucleic acids, for manipulating the resistance of plants topowdery mildew (Erysiphe cichoracearum). It further relates to plantswhich have been modified using such methods and materials.

PRIOR ART

[0002] Plant disease resistance (R) genes couple the recognition ofspecific pathogens to the induction of broad-spectrum defences thatrestrict the invader at the point of infection(1, 2). Manyplant-pathogen interactions conform to the gene-for-gene model whichpredicts that disease will develop if the infected plant lacks an R genefor recognition of the pathogen, or if the pathogen lacks thecorresponding (Avr) gene required for its recognition by the plant(3).The final outcome of a matched R-Avr interaction is incompatibility.

[0003] More than twenty plant R genes have been cloned andcharacterised. These are represented by proteins having fivecombinations of domains for a coiled-coil (CC)(4), leucine rich repeats(LRRs) (5), a transmembrane (TM) region, a protein kinase, a nucleotidebinding site (NBS), and with similarity to the Toll/interleukin receptor(TIR) (3). With the exception of Pto, which is a protein kinase, allcharacterised R genes contain LRRs. The eight R genes characterised inArabidopsis thaliana belong to the CC-NBS-LRR and TIR-NBS-LRRclasses(4), and a further 200-300 homologues of these are predicted inits genome(6).

[0004] The characterisation and cloning of R genes, particularly thosehaving novel structures, specificities or recognitions, allows thepathogen resistance traits arising from those genes to be manipulated.This is particularly important when dealing with commerciallysignificant pests.

[0005]A. thaliana has been used as a model to study genes for resistanceto powdery mildews, which cause severe losses on a wide range of cropspecies (7). Resistance of A. thaliana accession Ms-0 to the powderymildew pathogen Erysiphe cichoracearum isolate UCSC1 is regulated at theRESISTANCE TO POWDERY MILDEW8 (RPW8) locus on chromosome 3 (8). However,although this specificity had been defined in the prior art, the gene orgenes giving rise to the specificity had not been accurately mapped orcloned.

DISCLOSURE OF THE INVENTION

[0006] The present invention is based on the characterisation of novelRPW resistance genes from a cosmid (designated B6) prepared from agenomic library prepared from A. thaliana accession Ms-0, anddemonstrated to confer resistance to E. cichoracearum UCSC1 whentransferred to the susceptible accession, Col-0 (9).

[0007] Briefly, the present inventors had sought to isolate the gene forresistance at the RPW8 locus from cosmid B6, believing that it would beeither a TIR-NBS-LRR, or a CC-NBS-LRR gene. Interestingly, however,inspection of the DNA sequence in the DNA fragment B6 containing RPWBrevealed neither a TIR-NBS-LRR, nor a CC-NBS-LRR homologue. The DNAsequence of B6 revealed only a potential gene for a protein kinase,SKP-2, and two potential genes which were unrelated at the nucleotidesequence level and at the predicted protein sequence level, to any ofthe other characterised plant disease resistance genes, or indeed to anyother plant gene. These latter genes were named MSC1 and MSC2. Because atomato resistance gene, Pto, is protein kinase it was anticipated thatthe SKP-2/Ms-0 homologue might be RPW8. This was tested by makingsubclones containing differing regions of B6, and introducing these intoA. thaliana Col-0 by stable transformation. Unexpectedly, it was foundthat SK-2 did not confer resistance, but instead that MSC1 and MSC2independently conferred resistance to E. cichoracearum UCSC1, and tothree other powdery mildew diseases also. The existence of homologues inother plants has also been correlated with activity.

[0008] The genes MSC1 and MSC2 have therefore been designated RPW8.1 andRPW8.2, respectively.

[0009] The RPW8.1 and RPW8.2 proteins have 45.2% sequence identity, butare both relatively small and basic (pIs of greater than 9) and appearto contain both an N-terminal transmembrane (TM) domain (or possibly acleavage signal peptide) and a coiled coil (CC) domain. The proteinshave no significant similarity to the derived proteins from otherisolated or characterised R-genes, or indeed any plant gene, andtherefore appear to define a new class of R-gene product which isdesignated herein “TM-CC” class.

[0010] Thus in a first aspect of the present invention there isdisclosed a nucleic acid molecule encoding a plant resistance gene ofthe TM-CC class. Nucleic acid molecules according to the presentinvention may be provided isolated and/or purified from their naturalenvironment, in substantially pure or homogeneous form, or free orsubstantially free of other nucleic acids of the species of origin.Where used herein, the term “isolated” encompasses all of thesepossibilities.

[0011] The nucleic acid molecules may be wholly or partially synthetic.In particular they may be recombinant in that nucleic acid sequenceswhich are not found together in nature (do not run contiguously) havebeen ligated or otherwise combined artificially. Alternatively they mayhave been synthesised directly e.g. using an automated synthesiser.

[0012] The resistance genes of the invention will generally be powderymildew resistance genes, by which is meant a gene encoding a polypeptidecapable of recognising and activating a defense response in a plant inresponse to challenge with a powdery mildew pathogen, such as any of the15 isolates of E. cichoracearum tested herein; E. cruciferarum isolateUEA1; E. orontii isolate MGH; Oidium lycopersici isolate Oxford, or ineach case an elicitor thereof.

[0013] As will be well understood by those skilled in the art,“resistance” should not be taken to require complete resistance toinfection, but may in some cases be manifest as a reduced susceptibilityto the pathogen in question as compared to a control plant. Preferablythe resistance response is a specific response, in that (for instance)the gene will not provide resistance against other pathogens e.g. downymildew fungus P. parasitica Noco2.

[0014] The activity of the encoded polypeptide may be tested, forinstance, by challenging a plant in which the corresponding gene hasbeen introduced.

[0015] Plants to which the invention may be most advantageously appliedinclude any which are susceptible to powdery mildew.

[0016] Nucleic acid according to the present invention may include cDNA,RNA, genomic DNA and modified nucleic acids or nucleic acid analogs.Where a DNA sequence is specified, e.g. with reference to a figure,unless context requires otherwise the RNA equivalent, with U substitutedfor T where it occurs, is encompassed.

[0017] Complement sequences of those discussed herein are alsoencompassed. As is well understood by those skilled in the art, twonucleic acid nucleotide sequences are “complementary” when one willproperly base pair with all or part of the other according to thestandard rules (G pairs with C, and A pairs with T). One sequence is“the complement” of another where those sequences are of the samelength, but are complementary to each other.

[0018] Preferably the gene is derived from the RPW8 locus, for instancein Arabidopsis thaliana Ms-0. However, as described below, the work doneby the present inventors suggests that this locus may in fact beidentical with the RPW7 locus (which controls resistance to E.cruciferarum). Genes of this type have also been found by the presentinventors in other accessions and other species.

[0019] Thus in one embodiment of this aspect of the invention, there isdisclosed a nucleic acid comprising an RPW8.1 or RPW8.2 sequence, whichare described in Sequence Listing 2 below, which details thecomplementary nucleotides that define the transcription start, the firstexon, the intron and the second exon, and the transcription end.Sequences which are degeneratively equivalent to the coding sequences(encode the same polypeptide) are, of course, also embraced. Thus anucleic acid of the present invention may also be any which encodes anamino acid sequence (based on exon 1 and exon 2) of the RPW8. 1 orRPW8.2 sequences which are described in Sequence Listing 2 below. Theseare also listed in FIG. 2.

[0020] Further RPW8.1 or RPW8.2 sequences, from a variety of Arabidopsisaccessions, are shown in the sequence lineups hereinafter.

[0021] In a further aspect of the present invention there are disclosednucleic acids which are variants (including alleles, homologues,orthologues, mutants and derivatives) of the sequences of the firstaspect.

[0022] A variant nucleic acid molecule shares homology with, or isidentical to, all or part of the coding sequence discussed above.Generally, variants encode, or be used to isolate or amplify nucleicacids which encode, polypeptides which are capable of mediating aresponse against a pathogen, particularly powdery mildew.

[0023] Variants of the present invention can be artificial nucleic acids(i.e. containing sequences which have not originated naturally) whichcan be prepared by the skilled person in the light of the presentdisclosure. Artificial variants (derivatives) may be prepared by thoseskilled in the art, for instance by site directed or random mutagenesis,or by direct synthesis. Preferably the variant nucleic acid is generatedeither directly or indirectly (e.g. via one or amplification orreplication steps) from an original nucleic acid having all or part ofthe sequences of the first aspect. Preferably it encodes a powderymildew resistance gene.

[0024] Alternatively they may be novel, naturally occurring, nucleicacids, isolatable using the sequences of the present invention (e.g.those found in other A. thaliana accessions, or other plant species, asdescribed hereinafter). Sequence variants which occur naturally may alsoinclude alleles (which will include polymorphisms or mutations at one ormore bases).

[0025] Examples are shown e.g. in Sequence listing 1 which includesthree RPW8 homologues HR1, HR2, HR3 from A. thaliana accession Ms-0.

[0026] Thus a variant may be or include a distinctive part or fragment(however produced) corresponding to a portion of the sequence provided.These portions may include motifs which are distinctive to RPW8sequences, such motifs being discussed below in relation to primers.Preferred sequences are those which include the DIKE motif.

[0027] Fragments may encode or omit particular functional parts of thepolypeptide, e.g. CC or TM regions. Equally the fragments may haveutility in probing for, or amplifying, the sequence provided or closelyrelated ones. Suitable lengths of fragment, and conditions, for suchprocesses are discussed in more detail below. Also included are nucleicacids which have been extended at the 3′ or 5′ terminus with respect tothose of the first aspect.

[0028] The term ‘variant’ nucleic acid as used herein encompasses all ofthese possibilities. When used in the context of polypeptides orproteins it indicates the encoded expression product of the variantnucleic acid.

[0029] Some of the aspects of the present invention relating to variantswill now be discussed in more detail.

[0030] Homology (either similarity or identity) may be as defined anddetermined by the TBLASTN program, of Altschul et al. (1990) J. Mol.Biol. 215: 403-10, which is in standard use in the art, or, and this maybe preferred, the standard program BestFit, which is part of theWisconsin Package, Version 8, September 1994, (Genetics Computer Group,575 Science Drive, Madison, Wis., USA, Wisconsin 53711). BestFit makesan optimal alignment of the best segment of similarity between twosequences. Optimal alignments are found by inserting gaps to maximizethe number of matches using the local homology algorithm of Smith andWaterman.

[0031] Homology, with respect to either RPW8.1 or 8.2 or both, may be atthe nucleotide sequence and/or encoded amino acid sequence level.Preferably, the nucleic acid and/or amino acid sequence shares at leastabout 50%, or 60%, or 70%, or 80% homology, most preferably at leastabout 90%, 95%, 96%, 97%, 98% or 99% homology.

[0032] Thus a variant polypeptide in accordance with the presentinvention may include within an amino acid sequences described herein asingle amino acid or 2, 3, 4, 5, 6, 7, 8, or 9 changes, about 10, 15,20, 30, 40 or 50 changes, or greater than about 50, 60, 70, 80 changes.In addition to one or more changes within the amino acid sequence shown,a variant polypeptide may include additional amino acids at theC-terminus and/or N-terminus.

[0033] Thus in a further aspect of the invention there is disclosed amethod of producing a derivative nucleic acid comprising the step ofmodifying the coding sequence of a nucleic acid comprising any one thesequences discussed above.

[0034] Changes to a sequence, to produce a derivative, may be by one ormore of addition, insertion, deletion or substitution of one or morenucleotides in the nucleic acid, which may lead to the addition,insertion, deletion or substitution of one or more amino acids in theencoded polypeptide. Changes may be desirable for a number of reasons,including introducing or removing the following features: restrictionendonuclease sequences; codon usage; other sites which are required forpost translation modification; cleavage sites in the encodedpolypeptide; motifs in the encoded polypeptide (e.g. binding sites).Leader or other targeting sequences (e.g. the putative TM region) may beadded or removed from the expressed protein to determine its locationfollowing expression. All of these may assist in efficiently cloning andexpressing an active polypeptide in recombinant form (as describedbelow).

[0035] Other desirable mutation may be random or site directedmutagenesis in order to alter the activity (e.g. specificity) orstability of the encoded polypeptide. Changes may be by way ofconservative variation, i.e. substitution of one hydrophobic residuesuch as isoleucine, valine, leucine or methionine for another, or thesubstitution of one polar residue for another, such as arginine forlysine, glutamic for aspartic acid, or glutamine for asparagine. As iswell known to those skilled in the art, altering the primary structureof a polypeptide by a conservative substitution may not significantlyalter the activity of that peptide because the side-chain of the aminoacid which is inserted into the sequence may be able to form similarbonds and contacts as the side chain of the amino acid which has beensubstituted out. This is so even when the substitution is in a regionwhich is critical in determining the peptides conformation. Alsoincluded are variants having non-conservative substitutions. As is wellknown to those skilled in the art, substitutions to regions of a peptidewhich are not critical in determining its conformation may not greatlyaffect its activity because they do not greatly alter the peptide'sthree dimensional structure.

[0036] In regions which are critical in determining the peptidesconformation or activity such changes may confer advantageous propertieson the polypeptide. Indeed, changes such as those described above mayconfer slightly advantageous properties on the peptide e.g. alteredstability or specificity.

[0037] Other methods for generating novel specificities may includemixing or incorporating sequences from related resistance genes into thesequences disclosed herein. For example restriction enzyme fragments ofrelated genes could be ligated together. An alternative strategy formodifying RPW sequences would employ PCR as described below (Ho et al.,1989, Gene 77, 51-59) or DNA shuffling (Crameri et al., 1998, Nature391).

[0038] In a further aspect of the present invention there is provided amethod of detecting, identifying and/or cloning (isolating) a nucleicacid of the present invention (e.g. a homologue of the sequences set outhereinafter) from a plant which method employs any of the sequences ofthe invention discussed above. In particular the methods will generallyemploy primers or probes derived from all or part of these sequences (orsequences complementary thereto) set out herein. Preferably the plant isa species other than Arabidopsis.

[0039] An oligonucleotide primer for use in amplification reactions maybe about 30 or fewer nucleotides in length. Generally specific primersare upwards of 12, 13, 14, 15, 18, 21 or 24 nucleotides in length. Foroptimum specificity and cost effectiveness, primers of 16-24 nucleotidesin length may be preferred.

[0040] An oligonucleotide or polynucleotide probe may be based on theany of the sequences disclosed herein (e.g. introns or exons, althoughthe latter may be preferred). If required, probing can be done withentire restriction fragments of the genes which may be 100's or even1000's of nucleotides in length.

[0041] Those skilled in the art are well versed in the design of primersfor use processes such as PCR. The primers will usually be based onsequences which are peculiar or unique to the RPW sequences.Particularly preferred are the primers set out in any of the Examplesshown below. Primers based on the TM or CC regions may also bepreferred. Indeed, primers of the invention may be any of those whichoccur to the skilled person in the light of the disclosure herein, andin particular the sequence lineups shown hereinafter. For instancereferring to the cDNA nucleotide sequence of RPW8.1 from Ms-0 whenaligned with that of RPW8.1 homologues isolated from other A. thalianaaccessions, preferred primers may be based on e.g. the first 30nucleotides or so at the 5′ end, plus any conserved sequence near the 3′end (e.g. between 427 and 504 using the numbering given in the lineup).

[0042] Referring to the predicted amino acid sequence of RPW8.1 fromMs-0 as aligned with RPW8.1 homologues from other A. thalianaaccessions, degenerate primers may be based on any region within thefirst 30 amino acids or so, or (at the C-terminal) the conserved regionbetween 153 and 168.

[0043] One particularly preferred region for use in devising degenerateprimers is the DIKEIKAKISE motif at positions 142-152.

[0044] Referring to the cDNA nucleotide sequence of RPW8.2 from Ms-0, asaligned with that of RPW8.2 homologues isolated from other A. thalianaaccessions, primers may be devised particularly based on fully conservedregions near the 3′ and 5′ ends.

[0045] Finally, referring to the predicted amino acid sequence of RPW8.2from Ms-0, as aligned with RPW8.2 homologues isolated by PCR A. thalianaaccessions, preferred degenerate primers may be based on appropriatelyconserved regions therein e.g. encoding amino acids from the followingmotifs: MIAEVAAGGA LGLALSV; RLKLLLENAV SLVEENAELR RRNVRKKFRY MRDIKEFEAK;VDVQ VNQLADIKEL KAKMSEISTK LDK.

[0046] When using such probes or primers, if need be, clones orfragments identified in the search can be extended. For instance if itis suspected that they are incomplete, the original DNA source (e.g. aclone library, mRNA preparation etc.) can be revisited to isolatemissing portions e.g. using sequences, probes or primers based on thatportion which has already been obtained to identify other clonescontaining overlapping sequence.

[0047] In one embodiment, nucleotide sequence information providedherein may be used in a data-base (e.g. of expressed sequence tags, orsequence tagged sites) search to find homologous sequences, such asthose which may become available in due course, and expression productsof which can be tested for activity as described below.

[0048] In a further embodiment, a variant in accordance with the presentinvention is also obtainable by means of a method which includes:

[0049] (a) providing a preparation of nucleic acid, e.g. from plantcells,

[0050] (b) providing a probe or primer as discussed above,

[0051] (c) contacting nucleic acid in said preparation with said nucleicacid molecule under conditions for hybridisation of said nucleic acidmolecule to any said gene or homologue in said preparation, andidentifying said gene or homologue if present by its hybridisation withsaid nucleic acid molecule.

[0052] Plants which may be a suitable source of RPW8 may include any ofthose which may be susceptible to powdery mildew. For instance, thepowdery mildew fungus E. cichoracearum UCSC1 causes disease in a widerange of plant species, including members of the Cruciferae (e.g.Arabidopsis thaliana) Solanaceae (e.g. Lycopersicon esculentum (tomato),and Nicotiana spp (tobacco)) and Cucurbitaceae (e.g. squash).

[0053] Preferred plants for use in the present invention may thereforeinclude Crucifers (such as oil seed rape, broccolis, cauliflowers,cabbages, curly kale and the like), members of Solanaceae which areaffected by powdery mildew (e.g. tomato and tobacco), members ofCucurbitaceae (e.g. squash) and monocots (such as barley and wheat).Specific examples of methodologies used with some of these species areset out hereinafter). It is noted that even plants which are susceptibleto certain powdery mildew isolates may be a source of sequence which isuseful e.g. against other isolates, or when present as a heterologoussequence in a different genetic background (for instance in a transgenicplant).

[0054] Probing may employ the standard Southern blotting technique. Forinstance DNA may be extracted from cells and digested with differentrestriction enzymes. Restriction fragments may then be separated byelectrophoresis on an agarose gel, before denaturation and transfer to anitrocellulose filter. Labelled probe may be hybridised to the DNAfragments on the filter and binding determined. DNA for probing may beprepared from RNA preparations from cells.

[0055] Test nucleic acid may be provided from a cell as genomic DNA,cDNA or RNA, or a mixture of any of these, preferably as a library in asuitable vector. If genomic DNA is used the probe may be used toidentify untranscribed regions of the gene (e.g. promoters etc.), suchas is described hereinafter. Probing may optionally be done by means ofso-called ‘nucleic acid chips’ (see Marshall & Hodgson (1998) NatureBiotechnology 16: 27-31, for a review).

[0056] Preliminary experiments may be performed by hybridising under lowstringency conditions. For probing, preferred conditions are those whichare stringent enough for there to be a simple pattern with a smallnumber of hybridisations identified as positive which can beinvestigated further.

[0057] For instance, screening may initially be carried out underconditions, which comprise a temperature of about 37° C. or less, aformamide concentration of less than about 50%, and a moderate to lowsalt (e.g. Standard Saline Citrate (‘SSC’)=0.15 M sodium chloride; 0.15M sodium citrate; pH 7) concentration.

[0058] Alternatively, a temperature of about 50° C. or less and a highsalt (e.g. ‘SSPE’=0.180 mM sodium chloride; 9 mM disodium hydrogenphosphate; 9 mM sodium dihydrogen phosphate; 1 mM sodium EDTA; pH 7.4).Preferably the screening is carried out at about 37° C., a formamideconcentration of about 20%, and a salt concentration of about 5×SSC, ora temperature of about 50° C. and a salt concentration of about 2×SSPE.These conditions will allow the identification of sequences which have asubstantial degree of homology (similarity, identity) with the probesequence, without requiring the perfect homology for the identificationof a stable hybrid.

[0059] Suitable conditions include, e.g. for detection of sequences thatare about 80-90% identical, hybridization overnight at 42° C. in 0.25MNa₂HPO₄, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55°C. in 0.1×SSC, 0.1% SDS. For detection of sequences that are greaterthan about 90% identical, suitable conditions include hybridizationovernight at 65° C. in 0.25M Na₂HPO₄, pH 7.2, 6.5% SDS, 10% dextransulfate and a final wash at 60° C. in 0.1×SSC, 0.1% SDS.

[0060] It is well known in the art to increase stringency ofhybridisation gradually until only a few positive clones remain.Suitable conditions would be achieved when a large number of hybridisingfragments were obtained while the background hybridisation was low.Using these conditions nucleic acid libraries, e.g. cDNA librariesrepresentative of expressed sequences, may be searched. Those skilled inthe art are well able to employ suitable conditions of the desiredstringency for selective hybridisation, taking into account factors suchas oligonucleotide length and base composition, temperature and so on.

[0061] Binding of a probe to target nucleic acid (e.g. DNA) may bemeasured using any of a variety of techniques at the disposal of thoseskilled in the art. For instance, probes may be radioactively,fluorescently or enzymatically labelled. Other methods not employinglabelling of probe include amplification using PCR (see below) or RN'asecleavage. The identification of successful hybridisation is followed byisolation of the nucleic acid which has hybridised, which may involveone or more steps of PCR or amplification of a vector in a suitablehost.

[0062] In a further embodiment, hybridisation of nucleic acid moleculeto a variant may be determined or identified indirectly, e.g. using anucleic acid amplification reaction, particularly the polymerase chainreaction (PCR). PCR requires the use of two primers to specificallyamplify target nucleic acid, so preferably two nucleic acid moleculeswith sequences characteristic of are employed. Using RACE PCR, only onesuch primer may be needed (see “PCR protocols; A Guide to Methods andApplications”, Eds. Innis et al, Academic Press, New York, (1990)).

[0063] Thus a method involving use of PCR in obtaining nucleic acidaccording to the present invention may be carried out as describedabove, but using a pair of nucleic acid molecule primers useful in (i.e.suitable for) PCR.

[0064] The methods described above may also be used to determine thepresence of one of the nucleotide sequences of the present inventionwithin the genetic context of an individual plant, optionally atransgenic plant which may be produced as described in more detailbelow. This may be useful in plant breeding programmes e.g. to directlyselect plants containing alleles which are responsible for desirabletraits in that plant species, either in parent plants or in progeny (e.ghybrids, F1, F2 etc.). Thus use of the markers defined in the Examplesbelow, or markers which can be designed by those skilled in the art onthe basis the nucleotide sequence information disclosed herein, formsone part of the present invention.

[0065] Specific examples of homologous nucleic acids are those fromBrassica rapa discussed in more detail in the Examples below. Thesequence of the genomic DNA, and the predicted cDNA, is shown for oneeach in Sequence Listings 7,8 (BrHR1), 10, 11 (BrHR2), and 13, 14(BrHR3) respectively. These sequences are highly homologous to eachother (83-97% at amino acid level) and show 44-74% amino acid identityto AtRPW8.1, AtRPW8.2 and AtHR1-3. As above, the invention also embracesany nucleic acid encoding the respective amino acid sequences (SequenceListings 9, 12, 15) and so on.

[0066] As used hereinafter, unless the context demands otherwise, theterm “RPW nucleic acids” is intended to cover any of the nucleic acidsof the invention described above, including functional variants.

[0067] In one aspect of the present invention, the RPW nucleic aciddescribed above is in the form of a recombinant and preferablyreplicable vector.

[0068] “Vector” is defined to include, inter alia, any plasmid, cosmid,phage or Agrobacterium binary vector in double or single stranded linearor circular form which may or may not be self transmissible ormobilizable, and which can transform prokaryotic or eukaryotic hosteither by integration into the cellular genome or existextrachromosomally (e.g. autonomous replicating plasmid with an originof replication).

[0069] Specifically included are shuttle vectors by which is meant a DNAvehicle capable, naturally or by design, of replication in two differenthost organisms, which may be selected from actinomycetes and relatedspecies, bacteria and eucaryotic (e.g. higher plant, yeast or fungalcells).

[0070] A vector including nucleic acid according to the presentinvention need not include a promoter or other regulatory sequence,particularly if the vector is to be used to introduce the nucleic acidinto cells for recombination into the genome, such as the SE7.5construct shown in FIG. 3.

[0071] Preferably the nucleic acid in the vector is under the controlof, and operably linked to, an appropriate promoter or other regulatoryelements for transcription in a host cell such as a microbial, e.g.bacterial, or plant cell. The vector may be a bi-functional expressionvector which functions in multiple hosts. In the case of genomic DNA,this may contain its own promoter or other regulatory elements and inthe case of cDNA this may be under the control of an appropriatepromoter or other regulatory elements for expression in the host cell

[0072] By “promoter” is meant a sequence of nucleotides from whichtranscription may be initiated of DNA operably linked downstream (i.e.in the 3′ direction on the sense strand of double-stranded DNA).

[0073] “Operably linked” means joined as part of the same nucleic acidmolecule, suitably positioned and oriented for transcription to beinitiated from the promoter. DNA operably linked to a promoter is “undertranscriptional initiation regulation” of the promoter.

[0074] Thus this aspect of the invention provides a gene construct,preferably a replicable vector, comprising a promoter operatively linkedto a nucleotide sequence provided by the present invention. Generallyspeaking, those skilled in the art are well able to construct vectorsand design protocols for recombinant gene expression. Suitable vectorscan be chosen or constructed, containing appropriate regulatorysequences, including promoter sequences, terminator fragments,polyadenylation sequences, enhancer sequences, marker genes and othersequences as appropriate. For further details see, for example,Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al,1989, Cold Spring Harbor Laboratory Press.

[0075] Many known techniques and protocols for manipulation of nucleicacid, for example in preparation of nucleic acid constructs, mutagenesis(see above discussion in respect of variants), sequencing, introductionof DNA into cells and gene expression, and analysis of proteins, aredescribed in detail in Current Protocols in Molecular Biology, SecondEdition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosuresof Sambrook et al. and Ausubel et al. are incorporated herein byreference.

[0076] A preferred vector is the SE7.5 construct (FIG. 3) whichcomprises is a 7.5 kb sequence spanning RPW8.1 and RPW8.2 in thepBIN19-Plus binary vector (F. A. VAN ENGELEN, J. W. MOULTHOFF, A. J.CONNER, J. NAP, A. PEREIRA, AND W. J. STIKEMA. 1995. “pBINPLUS: ANIMPROVED PLANT TRANSFORMATION VECTOR BASED ON pBIN19”. TRANSGENICRESEARCH 4, 288-290.).

[0077] In one embodiment of this aspect of the present invention, thereis provided a gene construct, preferably a replicable vector, comprisingan inducible promoter operatively linked to a nucleotide sequenceprovided by the present invention.

[0078] The term “inducible” as applied to a promoter is well understoodby those skilled in the art. In essence, expression under the control ofan inducible promoter is “switched on” or increased in response to anapplied stimulus. The nature of the stimulus varies between promoters.Some inducible promoters cause little or undetectable levels ofexpression (or no expression) in the absence of the appropriatestimulus. Other inducible promoters cause detectable constitutiveexpression in the absence of the stimulus. Whatever the level ofexpression is in the absence of the stimulus, expression from anyinducible promoter is increased in the presence of the correct stimulus.

[0079] As shown in the Examples below, it is believed that the RPW8promoters provided by the present invention are inter alia wound- andSA-inducible.

[0080] Particular of interest in the present context are nucleic acidconstructs which operate as plant vectors. Specific procedures andvectors previously used with wide success upon plants are described byGuerineau and Mullineaux (1993) (Plant transformation and expressionvectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOSScientific Publishers, pp 121-148).

[0081] Suitable promoters which operate in plants include theCauliflower Mosaic Virus 35S (CaMV 35S). Other examples are disclosed atpg 120 of Lindsey & Jones (1989) “Plant Biotechnology in Agriculture”Pub. OU Press, Milton Keynes, UK. The promoter may be selected toinclude one or more sequence motifs or elements conferring developmentaland/or tissue-specific regulatory control of expression. Inducible plantpromoters include the ethanol induced promoter of Caddick et al (1998)Nature Biotechnology 16: 177-180.

[0082] If desired, selectable genetic markers may be included in theconstruct, such as those that confer selectable phenotypes such asresistance to antibiotics or herbicides (e.g. kanamycin, hygromycin,phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin,imidazolinones and glyphosate).

[0083] The present invention also provides methods comprisingintroduction of such a construct into a host cell, particularly a plantcell.

[0084] In a further aspect of the invention, there is disclosed a hostcell containing a heterologous nucleic acid or construct according tothe present invention, especially a plant or a microbial cell.

[0085] The term “heterologous” is used broadly in this aspect toindicate that the RPW nucleic acid in question has been introduced intosaid cells of the plant or an ancestor thereof, using geneticengineering, i.e. by human intervention. A heterologous gene may replacean endogenous equivalent gene, i.e. one which normally performs the sameor a similar function, or the inserted sequence may be additional to theendogenous gene or other sequence.

[0086] Nucleic acid heterologous to a plant cell may be non-naturallyoccurring in cells of that type, variety or species. Thus theheterologous nucleic acid may comprise a coding sequence of or derivedfrom a particular type of plant cell or species or variety of plant,placed within the context of a plant cell of a different type or speciesor variety of plant. A further possibility is for a nucleic acidsequence to be placed within a cell in which it or a homolog is foundnaturally, but wherein the nucleic acid sequence is linked and/oradjacent to nucleic acid which does not occur naturally within the cell,or cells of that type or species or variety of plant, such as operablylinked to one or more regulatory sequences, such as a promoter sequence,for control of expression.

[0087] The host cell (e.g. plant cell) is preferably transformed by theconstruct, which is to say that the construct becomes established withinthe cell, altering one or more of the cell's characteristics and hencephenotype e.g. with respect to powdery mildew resistance.

[0088] Nucleic acid can be transformed into plant cells using anysuitable technology, such as a disarmed Ti-plasmid vector carried byAgrobacterium exploiting its natural gene transfer ability (EP-A-270355,EP-A-0116718, NAR 12(22) 8711-87215 1984), particle or microprojectilebombardment (U.S. Pat. No. 5,100,792, EP-A-444882, EP-A-434616)microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green etal. (1987) Plant Tissue and Cell Culture, Academic Press),electroporation (EP 290395, WO 8706614 Gelvin Debeyser) other forms ofdirect DNA uptake (DE 4005152, WO 9012096, U.S. Pat. No. 4,684,611),liposome mediated DNA uptake (e.g. Freeman et al. Plant Cell Physiol.29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U.S.A. 87:1228 (1990d) Physical methods for the transformation of plant cells arereviewed in Oard, 1991, Biotech. Adv. 9: 1-11.

[0089] Agrobacterium transformation is widely used by those skilled inthe art to transform dicotyledonous species. Recently, there has alsobeen substantial progress towards the routine production of stable,fertile transgenic plants in almost all economically relevant monocotplants (see e.g. Hiei et al. (1994) The Plant Journal 6, 271-282)).Microprojectile bombardment, electroporation and direct DNA uptake arepreferred where Agrobacterium alone is inefficient or ineffective.Alternatively, a combination of different techniques may be employed toenhance the efficiency of the transformation process, eg bombardmentwith Agrobacterium coated microparticles (EP-A-486234) ormicroprojectile bombardment to induce wounding followed byco-cultivation with Agrobacterium (EP-A-486233).

[0090] The particular choice of a transformation technology will bedetermined by its efficiency to transform certain plant species as wellas the experience and preference of the person practising the inventionwith a particular methodology of choice. It will be is apparent to theskilled person that the particular choice of a transformation system tointroduce nucleic acid into plant cells is not essential to or alimitation of the invention, nor is the choice of technique for plantregeneration.

[0091] Thus a further aspect of the present invention provides a methodof transforming a plant cell involving introduction of a construct asdescribed above into a plant cell and causing or allowing recombinationbetween the vector and the plant cell genome to introduce a nucleic acidaccording to the present invention into the genome.

[0092] The invention further encompasses a host cell transformed withnucleic acid or a vector according to the present invention especially aplant or a microbial cell. In the transgenic plant cell (i.e. transgenicfor the nucleic acid in question) the transgene may be on anextra-genomic vector or incorporated, preferably stably, into thegenome. There may be more than one heterologous nucleotide sequence perhaploid genome.

[0093] Generally speaking, following transformation, a plant may beregenerated, e.g. from single cells, callus tissue or leaf discs, as isstandard in the art. Almost any plant can be entirely regenerated fromcells, tissues and organs of the plant. Available techniques are reviewdin Vasil et al., Cell Culture and Somatic Cell Genetics of Plants, VolI, II and III, Laboratory Procedures and Their Applications, AcademicPress, 1984, and Weissbach and Weissbach, Methods for Plant MolecularBiology, Academic Press, 1989.

[0094] The generation of fertile transgenic plants has been achieved inthe cereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto,K. (1994) Current Opinion in Biotechnology 5, 158-162.; Vasil, et al.(1992) Bio/Technology 10, 667-674; Vain et al., 1995, BiotechnologyAdvances 13 (4): 653-671; Vasil, 1996, Nature Biotechnology 14 page702).

[0095] Plants which include a plant cell according to the invention arealso provided.

[0096] Plants in which it may be desirable to introduce RPW8 include anyof those discussed herein which are susceptible to any powdery midews.The powdery mildews that affect wheat and barley are Blumeria graminisf.sp tritici and Blumeria graminis f.sp hordei, respectively, while thepowdery mildew that affects tomato is Oidium lycopersici, which is alsoa pathogen of Arabidopsis, and is controlled by the RPW8 locus (asdescribed elsewhere in this document). Transgenic plants containingheterologous RPW8.1 and RPW8.2 can be tested for resistance to theappropriate powdery mildew pathogen.

[0097] In addition to the regenerated plant obtainable by the abovemethod, the present invention embraces all of the following: a clone ofsuch a plant; selfed or hybrid progeny; descendants (e.g. F1 and F2descendants) and any part of any of these. The invention also provides aplant propagule from such plants, that is any part which may be used inreproduction or propagation, sexual or asexual, including cuttings, andso on. In each case these embodiments will include a heterologous RPWnucleic acid according to the present invention.

[0098] The invention further provides a method of influencing oraffecting the degree of resistance of a plant to a pathogen,particularly powdery mildew, more particularly to one of the isolatesdiscussed above, the method including the step of causing or allowingexpression of a heterologous nucleic acid sequence as discussed abovewithin the cells of the plant.

[0099] The step may be preceded by the earlier step of introduction ofthe nucleic acid into a cell of the plant or an ancestor thereof.

[0100] The foregoing discussion has been generally concerned with usesof the nucleic acids of the present invention for production offunctional RPW polypeptides in a plant, thereby increasing its pathogenresistance. However the information disclosed herein may also be used toreduce the activity or levels of such polypeptides in cells in which itis desired to do so. For instance the sequence information disclosedherein may be used for the down-regulation of expression of genes e.g.using anti-sense technology (see e.g. Bourque, (1995), Plant Science105, 125-149); sense regulation [co-suppression] (see e.g. Zhang et al.,(1992) The Plant Cell 4, 1575-1588). Further options for down regulationof gene expression include the use of ribozymes, e.g. hammerheadribozymes, which can catalyse the site-specific cleavage of RNA, such asmRNA (see e.g. Jaeger (1997) “The new world of ribozymes” Curr OpinStruct Biol 7:324-335. Nucleic acids and associated methodologies forcarrying out down-regulation (e.g. complementary sequences) form onepart of the present invention.

[0101] The present invention also encompasses the expression product ofany of the functional nucleic acid sequences disclosed above, plus alsomethods of making the expression product by expression from encodingnucleic acid therefore under suitable conditions, which may be insuitable host cells.

[0102] Following expression, the recombinant product may, if required,be isolated from the expression system. Generally however thepolypeptides of the present invention will be used in vivo (inparticular in planta).

[0103] Purified RPW protein produced recombinantly by expression fromencoding nucleic acid therefor, may be used to raise antibodiesemploying techniques which are standard in the art. Antibodies andpolypeptides comprising antigen-binding fragments of antibodies form afurther part of the present invention, and may be used in identifyinghomologues from other plant species.

[0104] Methods of producing antibodies include immunising a mammal (e.g.mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or afragment thereof. Antibodies may be obtained from immunised animalsusing any of a variety of techniques known in the art, and might bescreened, preferably using binding of antibody to antigen of interest.

[0105] For instance, Western blotting techniques or immunoprecipitationmay be used (Armitage et al, 1992, Nature 357: 80-82). Antibodies may bepolyclonal or monoclonal.

[0106] Antibodies may be modified in a number of ways. Indeed the term“antibody” should be construed as covering any polypeptide having abinding domain with the required RPW specificity. Thus, this term coversantibody fragments, derivatives, functional equivalents and homologuesof antibodies, including any polypeptide comprising an immunoglobulinbinding domain, whether natural or synthetic. Chimaeric moleculescomprising an immunoglobulin binding domain, or equivalent, fused toanother polypeptide are therefore included. Cloning and expression ofChimaeric antibodies are described in EP-A-0120694 and EP-A-0125023. Ithas been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the V1 and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341, 544-546 (1989) which consistsof a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston etal, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fvdimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; PHolliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993).

[0107] Candidate RPW polypeptides may be screened using theseantibodies—e.g. by screening the products of an expression librarycreated using nucleic acid derived from an plant of interest, or theproducts of a purification process from a natural source. A polypeptidefound to bind the antibody may be isolated and then may be subject toamino acid sequencing. Any suitable technique may be used to sequencethe polypeptide either wholly or partially (for instance a fragment ofthe polypeptide may be sequenced). Amino acid sequence information maybe used in obtaining nucleic acid encoding the polypeptide, for instanceby designing one or more oligonucleotides (e.g. a degenerate pool ofoligonucleotides) for use as probes or primers in hybridization tocandidate nucleic acid, or by searching computer sequence databases, asdiscussed further below.

[0108] The above description has generally been concerned with thecoding parts of the RPW genes and variants and products thereof. Alsoembraced within the present invention are untranscribed parts of thegene.

[0109] Thus a further aspect of the invention is a nucleic acid moleculeencoding the promoter of an RPW nucleic acid.

[0110] The present inventors have used northern analysis to show thattranscripts for RPW8.1 and RPW8.2 increase in abundance during theresistance reaction suggesting a possible role for the promoters intransduction of the resistance signal.

[0111] Referring to the Sequence listing, the promoter of RPW8.1 islocated in the region 15904 (start end) to 14719. That of RPW8.2 iswithin 16829 to 19087 (start end). These promoters appear to be woundand SA induced (but not JA induced).

[0112] Also embraced by the present invention is a promoter which is amutant, derivative, or other homolog of any of the RPW promotersdiscussed above which has promoter activity. For instance it may bedesirable to find minimal elements or motifs responsible for theresistance specific regulation. This can be done by using restrictionenzymes or nucleases to digest an appropriate nucleic acid molecule,followed by an appropriate assay to determine the sequence required.Nucleic acid comprising these elements or motifs forms one part of thepresent invention.

[0113] “Promoter activity” is used to refer to ability to initiatetranscription. The level of promoter activity is quantifiable forinstance by assessment of the amount of mRNA produced by transcriptionfrom the promoter or by assessment of the amount of protein productproduced by translation of mRNA produced by transcription from thepromoter. The amount of a specific mRNA present in an expression systemmay be determined for example using specific oligonucleotides which areable to hybridise with the mRNA and which are labelled or may be used ina specific amplification reaction such as the polymerase chain reaction.

[0114] Use of a reporter gene facilitates determination of promoteractivity by reference to protein production. The reporter genepreferably encodes an enzyme which catalyses a reaction which produces adetectable signal, preferably a visually detectable signal, such as acoloured product. Many examples are known, including β-galactosidase andluciferase. Those skilled in the art are well aware of a multitude ofpossible reporter genes and assay techniques which may be used todetermine promoter activity. Any suitable reporter/assay may be used andit should be appreciated that no particular choice is essential to or alimitation of the present invention.

[0115] In a further aspect of the invention there is provided a nucleicacid construct, preferably an expression vector, including an RPWpromoter region or fragment, mutant, derivative or other homolog orvariant thereof having promoter activity, operably linked to aheterologous gene, e.g. a coding sequence, which is preferably not thecoding sequence with which the promoter is operably linked in nature.

[0116] The invention will now be further described with reference to thefollowing non-limiting Figures and Examples. Other embodiments of theinvention will occur to those skilled in the art in the light of these.

[0117] Sequences and Figures

[0118] Sequence listing 1: 27689 bp contiguous genomic sequence ofArabidopsis thaliana accession Ms-0 containing RPW8.1, RPW8.2, is threeRPW8 homologues HR1, HR2, HR3, and the Ms-0 homologue of SKP-2.Nucleotide locations are also given of the genomic constructs used fortransformation referred to in FIG. 1e: SE14: 4160-18466; CC7:6508-13551; SS10: 8718-18466; EE6.2: 12297-18466; EP3.7: 12297-16033;XE3.8: 14658-18466. Annotations give (where available), mRNA, codingsequence (CDS), exons, intron, transcription start, transcription end,protein sequence.

[0119] Sequence listing 2: Nucleotides 13801-18466 representing part ofthe DNA sequence of cosmid B6, numbered from the telomere end at markerB9 (FIG. 1a) and represents part of the sequence in Sequence listing 1.The sequence below contains only the two genes (mRNA) RPW8.1 and RPW8.2which individually control resistance to powdery mildew caused byErysiphe cichoracearum isolate UCSC1 and other powdery mildew pathogens.Annotations give, for each gene, the complementary nucleotides thatdefine the transcription start (determined by 5′RACE), the first exon,the intron and the second exon (determined by comparison of genomic andcDNA sequence), and the transcription end (determined by 3′ RACE). Thegiven protein coding sequence (CDS) sequence is the predicted amino acidtranslation of coding sequences in exon 1 and exon 2, for each gene.

[0120] Sequence listing 3: The cDNA nucleotide sequence of RPW8.1 fromMs-0 is aligned with that of RPW8.1 homologues isolated by PCR fromother A. thaliana accessions. Accessions resistant to Erysiphscichoracearum UCSC1 are Ms (=Ms-0), Wa (=Wa-1), Kas (=Kas-1) and C24(=C24). Accessions susceptible to E. cichoracearum UCSC1 were Can(=Can-0), Nd (=Nd), Sy (=Sy) and Ws (=Ws-0). Nucleotide differences arein bold. In this, and other searches, parameters used were default(Blosum=62, Gap penalty=11; per residue gap cost=1; lambda ratio=0.85)/

[0121] Sequence listing 4: The predicted amino acid sequence of RPW8.1from Ms-0 is aligned with RPW8.1 homologues isolated by PCR from otherA. thaliana accessions. Dash (-) indicates identity with the RPW8.1/Ms-0sequence; dot (.) indicates gap, or no equivalent sequence. Singleletter codes beneath the Ms-0 sequence indicate predicted differences.Accessions resistant to Erysiphs cichoracearum UCSC1 are Ms (=Ms-0), Wa(=Wa-1), Kas (=Kas-1) and C24 (=C24). Accessions susceptible to E.cichoracearum UCSC1 were Can (=Can-0), Nd (=Nd), Sy (=Sy) and Ws(=Ws-0). The analysis shows that the predicted amino acid sequence atRPW8.1 of the resistant accessions is identical to that of accessionMs-0. Susceptible accessions have amino acids different from theresistant accessions at one or more of the following positions: 31, 33,40, 43, 45, 77, 95, 108, an insertion at 121-141, and at 169.

[0122] Sequence listing 5: The cDNA nucleotide sequence of RPW8.2 fromMs-0 is aligned with that of RPW8.2 homologues isolated by PCR fromother A. thaliana accessions. Accessions resistant to Erysiphscichoracearum UCSC1 are Ms (=Ms-0), Wa (=Wa-1), Kas (=Kas-1) and C24(=C24). Accessions susceptible to E. cichoracearum UCSC1 were Can(=Can-0), Nd (=Nd), Sy (=Sy) and Ws (=Ws-0). Nucleotide differences arein bold. Stop codons are in italics.

[0123] Sequence listing 6: The predicted amino acid sequence of RPW8.2from Ms-0 is aligned with RPW8.2 homologues isolated by PCR from otherA. thaliana accessions. Dash (-) indicates identity with the RPW8.1/Ms-0sequence; dot (.) indicates gap, or no equivalent sequence. Singleletter codes beneath the Ms-0 sequence indicate predicted differences.Accessions resistant to Erysiphs cichoracearum UCSC1 are Ms (=Ms-0), Wa(=Wa-1), Kas (=Kas-1) and C24 (=C24). Accessions susceptible to E.cichoracearum UCSC1 were Can (=Can-0), Nd (=Nd), Sy (=Sy) and Ws(=Ws-0). The analysis shows that the predicted amino acid sequence atRPW8.2 of the resistant accessions is identical to that of accessionMs-0. Susceptible accessions have amino acids different from theresistant accessions at one or more of the following positions: 17, 19,64, 70, 111, 116, termination at 144, and 161. It is notable thataccession C24, which is resistant, has an RPW8.2 sequence different tothat of the other resistant accessions, whereas the RPW8.1 amino acidsequence is identical to that of the resistant accessions (see Sequencelisting 4).

[0124] Sequence listings 7-9: sequences for BrHR1—genomic DNA, predictedis cDNA, and predicted encoded amino acid respectively.

[0125] Sequence listings 10-12: sequences for BrHR2—genomic DNA,predicted cDNA, and predicted encoded amino acid respectively.

[0126] Sequence listings 13-15: sequences for BrHR3—genomic DNA,predicted cDNA, and predicted encoded amino acid respectively.

[0127]FIG. 1: Map-based cloning of RPW8. [a] Order of molecular markersused in the fine-mapping of RPW8. Vertical broken lines link the geneticlocation of markers to their physical position on subcloned DNA;sequence identity was demonstrated by hybridisation. Figures in bracketsare the numbers of plants with recombinations between RPW8 and theindicated marker closer to the telomere (t) or centromere (c). [b, c]Aligned A thaliana Col-0 genomic DNA from (b) VAC and (c) 8AC cloneswhich hybridised to the indicated molecular markers genetically linkedto RPW8. [d] Alignment of cloned A. thaliana Ms-0 genomic DNA whichhybridised to the indicated genetic markers. “(+)” indicates that Col-Oplants transformed with the DNA were resistant, and “(−)” indicates theywere susceptible, to E. cichoracearum UCSC1. [e] Restriction sites ofcosmid 86 used for sub-cloning A. thaliana Ms-0 DNA. ORFs detected inthe B6 sequence are shown as thick lines in the subclones. Subclones aremarked (+) and (−) as for (d) f. Aligned Ms-D cDNAs. Cloned cDNAsexpressed under a constitutive viral promoter are marked (+) and (−) asfor (d).

[0128]FIG. 2. Analysis of the RPW8locus. [a] The RPW8 locus consisted offive tandemly linked homologues. [b, c] Predicted amino acid sequencesof (a) RPW8.1 and (b) RPW8.2 from accession Ms-O. Sequences in italicsare predicted to form transmembrane (TM) domains, or possibly signalpeptides. Sequences in bold are predicted to form coiled coils (CC).Lowercase letters above the sequence indicate the heptad repeats thatdefine coiled coils in which ‘a’ and ‘d’ are typically hydrophobic,while the other residues tend to be hydrophilic.

[0129]FIG. 3. The SE7.5 plant transformation vector described in Example8.

[0130]FIG. 4. Identification of RPW8 homologs in Brassica rapa (R) andB. oleracea (O).

[0131] A. One BAC filter B. rapa probed with AtRPW8.1 & 2 and AtHR1-3sequentially, showing the same clones hybridised to the probes.

[0132] B. DNA from positive BAC clones was digested with EcoRI andBamHI, separated in agarose gel, blotted to membrane, and probed to theDNA mixtures as in A.

EXAMPLES Example 1 Localisation of RPW8

[0133] Plasmid B6

[0134]A. thaliana accession Col-0 is susceptible and accession Ms-0 isresistant to infection by E. cichoracearum UCSC1.

[0135] This was confirmed by observation 10 days after inoculation(results not shown) after which time Col-0 supported growth ofsuperficial white mycelium whereas Ms-0 did not. Resistance of accessionMs-0 is controlled by the RPW8 locus which maps genetically to an 8.5 cMinterval, flanked by markers CDC2A and AFC1(8) (FIG. 1a). A populationof 1,500 F₂ plants from a cross between accessions Ms-0 and Ler(susceptible to E. cichoracearum UCSC1) was screened to detectindividuals with recombination break points between markers g19397 andCDC2A (FIG. 1a). The ninety-four recombinants recovered were used tofine-map RPW8. Their genotypes at the RPW8 locus were deduced by scoringF₃ progeny for resistance or susceptibility to E. cichoracearum UCSC1.Their genotypes at selected RFLP markers in the g19397 and CDC2Aintervalrevealed that Atpk41A co-segregated with RPW8, and that YAC ends8E1-R and 9D1-R flanked the RPW8 locus (FIG. 1b). Atpk41A, 8E1-R and9D1-R were used as hybridisation probes to screen the TAMU and IGF BAClibraries, which were re-screened with BAC ends from some of therecovered clones. Five of the isolated clones formed a ˜200 kbcontiguous, overlapping series of that spanned RPW8 (FIG. 1c). A genomiclibrary of the resistant accession Ms-0 was constructed and screened forclones that hybridised to markers 8E1-R, Atpk41A, 6I2-L, and 3B3-L. Fiverecovered clones formed a 45 kb contiguous series that spanned the RPW8locus (FIG. 2d). RPW8 was flanked by genetic markers B9 and 3B3-L, whichwere both located in cosmid B6 (FIG. 1d). The B6 insert was introducedinto the powdery mildew-susceptible accession Col-0 byAgrobacterium-mediated transformation(10). The transformed progeny,represented here by T-B6, were resistant to infection by E.cichoracearum UCSC1(results nor shown) whereas plants transformed withcosmids S5-1 and J4-2 were susceptible (not shown). This confirmed thatcosmid B6 contained RPW8.

[0136] To localise RPW8, cosmid B6 was sequenced and a variety offragments of cosmid B6 were sub-cloned in a plant transformation vectorand introduced into Col-0 plants by Agrobacterium-mediatedtransformation (FIG. 1e). The DNA sequence of B6 revealed three ORFs.

[0137] One had similarity (predicted amino acid sequence identity was100%) to the cDNA ATHPROKINA (GenBank Accession L05561) from whichmarker Atpk41A was derived, and which corresponds to the gene proteinkinase SPK-2 (GenBank Accession S56718) located in BAC T20E23 (GenBankAccession AL133363) from A. thaliana accession Col-0 (FIG. 1c). Wetherefore named this ORF SPK-2/M to denote it as the Ms-0 allele ofSPK-2.

[0138] ORFs MSC1, and MSC2 had no obvious alleles in the A. thalianaCol-0 sequence in T20E23 (FIG. 1f).

[0139] Plants transgenic for subclones SE14, SS10, EE6.2, EP3.7 andXE3.8 were resistant to E. cichoracearum UCSC1, whereas plantstransgenic for subclone CC7 were susceptible (FIG. 1e, Sequence listing1 gives the sequence for these fragments). The subclones that conferredresistance contained either ORF MSC1 (EP3.7), or ORF MSC2 (XE3.8), orboth of these (FIG. 1e). This indicated that RPW8 comprised twoindependently-acting genes, MSC1 and MSC2, which were therefore re-namedRPW8.1 and RPW8.2 respectively. The entire 18466 nt B6 sequence,containing SKP-2/M, RPW8.1 and RPW8.2, and part of the contiguoussequence of cosmid J2-4 (FIG. 1e) is given in Sequence listing 1.

[0140] cDNAs for RPW8.1 and RPW8.2, and for SKP-2/M as control, werecloned into a plant transformation vector under control of the highlyactive cauliflower mosaic virus 35S promoter, and introduced into Col-0plants by Agrobacterium-mediated transformation. Transgenic plantsT-35s::RPW8.1 and T-35s::RPW8.2, were resistant to E. cichoracearumUCSC1 whereas the transgenic plants T-35s::SKP-2 were susceptible(results not shown). We concluded that RPW8 contains two functionalgenes, RPW8.1 and RPW8.2, which are each sufficient for resistance to E.cichoracearum UCSC1. Sequence listing 2 gives the 4665 nucleotidesequence of A. thaliana accession Ms-0 DNA which contains the predictedpromoters and the transcribed sequences of RPW8.1 and RPW8.2.

Example 2 Characterisation of Specificity Controlled by RPW8

[0141] A range of pathogens virulent on A. thaliana accession Col-0 wereused to characterise the specificity of resistance controlled by RPW8.Transgenic plants T-B6, T-35s::RPW8.1 and T-35s::RPW8.2 were resistantto all of the tested powdery mildew pathogens. These included 15isolates of E. cichoracearum, and E. cruciferarum isolate UEA1, E.orontii isolate MGH, and Oidium lycopersici isolate Oxford, representingfour distinct species(11). These results indicate that RPW7, whichcontrols resistance to E. cruciferarum UEA1 and maps with RPW8 betweenmarkers CDC2A and AFC1(8) (FIG. 1a), may be identical to RPW8.1 andRPW8.2. Significantly, T-B6 plants were susceptible to other pathogens,including the fungus Peronospora parasitica Noco2 to which Ms-0 wasresistant (testing 7 days after inoculation for white sporagiophores,results not shown), the cauliflower mosaic virus, and to the bacteriumPseudomonas syringae pv tomato DC3000 (results not shown). Because noneof the powdery mildew pathogens we have tested could infect plantscontaining the RPW8 locus, we have no formal evidence of a gene-for-geneinteraction. RPW8.1 and RPW8.2 defined in Sequence listing 2 appear torepresent a special type of R-gene which controls “specific” resistanceto a broad group of the powdery mildew pathogens.

Example 3 RPW8 Homologues from other Accessions

[0142] RPW8.1 produced a 908 nt transcript with a single 197 nt intronand 444 nt of predicted coding sequence, and RPW8.2 produced a 926 nttranscript with a 128 intron and 522 of predicted coding sequence(Sequence listing 1 & 2).

[0143] We examined the sequences of RPW8.1 and RPW8.2 homologues inseven other A. thaliana accessions with different levels of resistanceto E. cichoracearum UCSC1. Accessions Kas-1 and Wa-1 are stronglyresistant(12, 13), and a major resistance gene in each has beengenetically mapped to the RPW8 locus (S. Somerville, personalcommunication). RPW8.1 and RPW8.2 homologues were amplified from Kas-1and Wa-1 by PCR, and the DNA sequences were identical to those of thecorresponding Ms-0 genes in Sequence listing 2. RPW8.1 and RPW8.2homologues were also amplified by PCR from the moderately susceptibleaccessions, Ler, Nd-0, and Ws-0. Their derived amino acid sequencesdiffered from those of the corresponding Ms-0 genes by 1.1-4.1%. Wecould not detect either an RPW8.1 or an RPW8.2 homologue in theextremely mildew-susceptible accession Col-0(13), by Southern analysis(results not shown), PCR, or by inspection of the published sequence atthe RPW8 locus in Col-0 (BAC 20E23, FIG. 1c). Resistance of A. thalianato E. cichoracearum UCSC1 in these A. thaliana accessions is thereforeassociated with extreme conservation of DNA sequence at RPW8.1 andRPW8.2.

Example 4 RPW8 Homologues on Cosmid B6

[0144] Southern blotting showed that RPW8.1 and RPW8.2 were present inMs-0 and Kas-1 as single-copy genes (not shown). However, the nucleotidesequence of cosmid B6 and J4-2 (FIG. 1c, Sequence listing 1) revealedthat RPW8 was linked to three ORFs with 55.0-64.2% DNA sequence identityto RPW8.1 and RPW8.2. These were named Homologous to RPW81 (HR1), HR2,and HR3, and they were also closely related (99.4-99.9% DNA sequenceidentity) to predicted genes CAB62476, -5 and -4, respectively, in BAC20E23 from accession Col-0. A recombination break-point between RPW8.2and HR3, detected with marker 3B3-L (FIG. 1a) indicated that HR1, -2,and -3 were not required for resistance to powdery mildew. The RPW8locus of Ms-0 therefore contains five tandemly arranged RPW8 homologues(FIG. 2a, annotated also in Sequence listing 1), three of which are alsorepresented in Col-0. Other R gene-loci also contain clusters ofhomologues(14), members of which may recognise different strains of thepathogen(15). These gene clusters apparently evolve new R-genespecificities rapidly, through duplication, unequal crossover andmutation(16, 17). A comparison of HR1, -2, -3, RPW8.1, and -2 by PILEUP,below, shows regions of similarity between the predicted proteins. {hr1}MPvsEimaGA ALGLaLQvLH dAikkAKDrS ltTrcILdRL dATIfrITPl {hr2} MPltEiiaGAALGLaLQiLH eAiqrAKDrS ltTscILdRL dsTIlrITPl {hr3} MP1vElltsA ALGLsLQlLHeAiirAKekt liTrcILdRL dATlhkITPf {rpw82} ˜miaEvaaGg ALGLaLsvLHeAvkrAKDrS vtTrfILhRL eATIdsITPl {rpw81} MPigElaiGA vLGvgaQaiydrfrkArDiS .....fvhRL cATIlsIePf Consens MP--E---GA ALGL-LQ-LH-A---AKD-S --T--IL-RL -ATI---ITP-51                                                 100 {hr1} vtqvDKlseEvedSp.RKVi EdLKhLLEkA vsLVEAYAEL rRRNlLkKfR {hr2} makveKlnkE sdeSl.RKVfEdLKhLLEkA vvLVEAYAEL kRRNlLeKyR {hr3} vikiDtlteE vdepf.RKVi EeLKrLLEkAirLVdAYAEL klRNlLrKyR {rpw82} vvqiDKfseE medStsRKVn krLKlLLEnAvsLVEenAEL rRRNvrkKfR {rpw81} lvqiDKrsk. vegSplReVn ErLtcfLElAyvfVEAYpkL rRRqvLrKyR Consens ----DK---E ---S--RKV- E-LK-LLE-A--LVEAYAEL -RRN-L-K-R101                                                150 {hr1} YkRrIKElEasLRWmvDVDV QVNQWvDIKe LmAKMSEMnT KLdeItrQP. {hr2} YkRrIKElEg sLkWmvDVDVkVNQWaDIKd LmAKMSENnT KLekImgQP. {hr3} YkRrIKElds sLRWmiDVDV QVNQWlDIKkLmgKMSEMnT KLddItrQP. {rpw82} YmRdIKEfEa kLRWvvDVDV QVNQlaDIKeLkAKMSEisT KLdkImpQPk {rpw8l} YikaIetiEl aLRsiivVDf QVdQWdDIKeikAKiSEMdT KLaevisacs Consens Y-R-IKE-E- -LRW--DVDV QVNQW-DIK-L-AKMSEM-T KL--I--QP-151                                                 200 {hr1} tdcicfksnhstsqsssqni veetdrslee ivecssdgsk pkidihihws {hr2} idciisedn. .....tnmdivervdpslea kagcsnsdsk pkidihlrws {hr3} .......... .......mdi ieatgrsseed.gc....tk ptidihfrw. {rpw82} feihigwcsg ktnrairftfcsdds˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜ {rpw81}kira˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜ Consens -------------------- ---------- ---------- ---------- 201          215 {hr1}srkrnkdrei rfvlk {hr2} ..kqskdhgi rfvln {hr3} .knqtkehei rfifk {rpw82}˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜ {rpw81} ˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜ Consens ---------- -----

[0145] HR1, -2, and -3 may therefore represent R-genes with as-yetunknown specificity. BLAST searches with these peptides show no obvioussimilarity to any other characterised gene products, however. Thissuggests that the RPW8 proteins represent a novel type of protein.

Example 5 Northern Analysis

[0146] Northern analysis indicated that transcripts for RPW8.1 andRPW8.2 of the appropriate size were expressed in uninoculated T-B6plants, but that transcript levels increased in abundance during theresistance reaction (not shown).

Example 6 Structure of RPW8.1 and 8.2

[0147] The predicted RPW8.1 and RPW8.2 proteins have 45.2% sequenceidentity, and are relatively small (molecular weights 17,000 and 19,973,respectively) and basic (pIs of 9.46 and 10.05, respectively). RPW8.1and RPW8.2 had no significant similarity to the derived proteins fromother R-genes, nor to any characterised plant gene. Analysis of the RPW8sequences indicated a predicted N-terminal TM domain, or possibly acleavage signal peptide, and a CC domain (FIGS. 2b & c). Therefore RPW8defines a new class of R-gene product, which we name here TM-CC.

Example 7 RPW8 Homologues from other Plant Species

[0148] Barley

[0149] In the light of the present disclosure, those skilled in the artwill appreciate that RPW8 homologues may be isolated from barley by anyof several techniques.

[0150] A preferred method is to identify clones in a genomic library ofbarley that hybridise to DNA for RPW8.1 and/or RPW8.2 as follows.

[0151] A cosmid library representing the barley genome might contain80,000 clones each with an insert size of 20-40 kb. These are storedindividually. DNA from each clone is isolated, and 40 pools are made,each containing DNA from 2,000 clones. Samples from the pools can bedigested with EcoRI, or another suitable enzyme which releases thevector sequence. Digested DNA samples are run out on 1% TAE agarosegels. The DNA in the gels is treated with standard depurination,denaturation and neutralisation buffer (Sambrook et al., 1989) beforeovernight capillary blotting onto Hybond N⁺ (Amersham) membrane with10×SSC, and fixation at 80° C. for 2 hours. DNA representing the codingregion of RPW8.1 and RPW8.2 is amplified from Arabidopsis thalianaaccession Ms-0 cosmid B6 by PCR with specific primers (such asGACCCGTACAGTACTAAGTCTA and GATTTCCGAAATTGATTACAAGAA for RPW8.1, and forRPW8.2, the primers AACTCTTCACCTCGAGAGCTAACA andAGTCGTTTGACACAATTGGGACAT), labelled with ³²P-dCTP with a Multiprime DNAlabelling kit (Amersham) according to the manufacturer's instructions.Blots are washed 2-3 times in 2×SSC, 0.1% SDS (low stringency wash) at65° C. and, if necessary, in 0.2% SSC, 0.1% SDS (high stringency wash).Hybridisation signal is detected by phosphor screens scanned in a Storm840 phosphor imager (Molecular Dynamics).

[0152] Pools that reveal bands where digested DNA has hybridised to theprobe DNA are then reconstituted as 40 sub-pools, each containing theDNA from 50 clones. The sub-pools are screened again with probes RPW8.1and RPW8.2, as described for the pools, and sub-pools that revealhybridising bands are identified. The chosen sub-pools are nowrepresented as DNA samples from each of the 50 constituent clones, andthese are screened as for the pools, to identify the genomic clone thatgave rise to an hybridising band in the pool, and in the sub-pool.

[0153] The process will therefore identify one or more clones of genomicDNA from barley that contain sequences homologous to the RPW8 genes. Toprecisely identify the barley DNA sequence that is homologous to RPW8,an efficient approach is to subclone the cosmid as 2-5 kb fragments in achosen vector, such as Bluescript. Subclones are then screened as colonyblots according to the manufacturer's instructions of the nitrocellulosemembrane, using 32-P-labelled RPW8.1 and RPW8.2 as probe. Individualsubclones that hybridize to the probes are recovered, and the cloned DNAis sequenced. Software programs such as Blast, and PileUp are used tolocate regions in the subcloned DNA with similarity to RPW8.1 andRPW8.2.

[0154]Brassica napus

[0155] Southern analysis was used to determine if sequences in Brassicanapus hybridized to RPW8.1 and RPW8.2. It was found that B. napus doescontain DNA which hybridized to RPW8.1 and RPW8.2, suggesting that RPW8homologues occur in this species.

[0156] Southern Analysis of Brassica napus

[0157] DNA was extracted from leaves of B. napus as follows.Approximately 3 g of leaves were ground into powder with liquid nitrogenin a pre-chilled mortar. The powder was transferred to a 50 mlcentrifuge tube and carefully mixed with 20 ml of urea extraction buffer(8 M urea, 50 mM Tris pH8, 20 mM EDTA pH 8, 350 mM NaCl, 2% sarcosineand 5% phenol). 800 microlitres 20% SDS was added and the mixture wasincubated at 65° C. for 10 min. The solution was extracted withphenol/chloroform (1:1), centrifuged at 2,000 g and the aqueous phasewas extracted again with phenol/chloroform (1:1), maintained at 4° C.for 20 min, 4 ml 5 mM potassium acetate was added, the samples gentlymixed and held on ice for 30 min. The debris was spun down at 2,000 g,4° C. for 20 min, and 16 ml isopropanol was gently mixed into thesupernatant. The DNA was immediately pelleted at 2000 g for 15 min. Thepellets were dried and then dissolved in 1 ml TE.

[0158] DNA was digested overnight with the restriction enzyme EcoRI.Digested DNA samples were separated on 1% TAE agarose gels. DNA in thegels was depurinated, denatured and neutralised (Sambrook et al., 1989),transferred to Hybond N⁺ (Amersham, UK) nylon membranes by capillaryblotting overnight with 10×SSC, and fixed to the membrane at 80° C. for2 hours.

[0159] RPW8.1 and RPW8.2 were amplified by PCR from genomic DNA ofArabidopsis thaliana accession Ms-0, using as primers the sequencesdesigned to against the beginning and the end of the predicted codingsequences. The amplified products were labelled with ³²P-dCTP using aMultiprime DNA labelling kit (Amersham, UK) according to themanufacturer's instructions. Blots were washed 2-3 times in 2×SSC, 0.1%SDS (low stringency wash) at 65° C. and, if necessary, in 0.2% SSC, 0.1%SDS (high stringency wash). Hybridisation was detected by phosphorscreens scanned in a Storm 840 phosphor imager (Molecular Dynamics,USA).

[0160] Two bands, 4 kb and 1 kb, could be distinguished in the lanes forB. napus.

[0161]Brassica rapa and B. oleracea

[0162] BAC libraries of Brassica rapa (B. rapa ssp oleifera cv R018) ofB. oleracea (B.oleracea ssp. alboglabra cv A12) were constructed.

[0163] These libraries were screened using a mixture of AtRPW8.1 andAtRPW8.2 genomic DNA (amplified with AtRPW8-specific primers describedabove with 6I2B6 cosmid DNA as template) as probe, with 50 ng of eachPCR product being mixed and used for the labelling with dCTP³².

[0164] Hybridisation was carried out at 50° C. overnight, and thefilters were washed at 50° C. with 2×SSC and 0.1% SDS solution threetime. Eighteen BAC clones from B. rapa and 7 clones B. oleracea fromwere identified hybridising to AtRPW8.

[0165] A second hybridisation with the same filters under the sameconditions mentioned above using as probe the DNA mixture of AtHR1,AtHR2 and AtHR3 (these were each amplified by primers corresponding tothe first 24 bp and last 24 bp of the predicted coding sequences ofthese three homologs—˜30 ng DNA from each was mixed for labelling)indicated the same BAC clones also hybridised with the AtHR genes (seeFIG. 4).

[0166] Fingerprinting was performed according to the manufacturer'sinstructions as follows: the DNA of all the BAC clones was digested withEcoRI and BamHI, separated on 0.8% agarose gel, and then photographedunder UV light following hybridisation. For the AtHR1-3 DNA probes thedigested DNA in the gel was blotted to a Nitrocellulose membranepurchased from Roche. The blots were sequentially probed withdCTP³²-labelled AtRPW8 and AtHR1-3 DNA mixtures described above underthe same conditions

[0167] Fingerprinting revealed that the genomes of both B. rapa and B.oleracea contain a single RPW8-like locus since all the positive BACclones from either B. rapa or B. oleracea formed only one overlappingcontig, as does the Arabidopsis genome (FIG. 2). Subcloning andsequencing was first performed with one positive BAC clone from B. rapa.

[0168] We found the B. rapa genome contains three RPW8-like genestandemly linked with each other. The sequence of the genomic DNA, thepredicted cDNA and deduced amino acids was listed in the sequencelisting.

[0169] These three genes (named BrHR1, BrHR2, and BrHR3) are highlyhomologous to each other (83-97% at amino acid level) and show 44-74%amino acid identity to AtRPW8.1, AtRPW8.2 and AtHR1-3.

[0170] Further results have shown that B. oleracea genome also containsthree RPW8-like sequences (named BoHR1, BoHR2, and BoHR3), and theorganisation of these genes is very similar to that of the B. rapahomologs.

[0171] It is clear that these three genes are the Brassica homologs ofAtRPW8 genes. Thus the AtRPW8.1 and AtRPW8.2 genomic DNA hybridises tothe 3 Brassica sequences, so does the AtHR1, AtHR2 and AtHR3 genomicDNA. Secondly, BLAST search shows that these 3 sequences only pickAtRPW8 and its homologs, and they show considerably high homology to theAtRPW8 family members. Thirdly, these 3 homologs are highly homologousto each other, and to AtHR3, implying they share a common origin.

[0172] Expression of the genes may be conformed by RT-PCR, while theirresistance function can be confirmed by putting them under the controlof AtRPW8 promoter(s) and introducing them into Arabidopsis Col-0background which is then challenged by the same pathogens discussedabove.

Example 8 Introduction of RPW8 Into Plant Species

[0173] Transformation of Nicotiana benthamiana with Cosmid B6

[0174] RPW8 was transferred to Nicotiana benthamiana by stable,Agrobacterium mediated transformation of N. benthamiana plants withcosmid B6. Rather surprisingly, the transgenic plants were resistant toE. cichoracearum. This indicated that RPW8 functioned in theheterologous host, N. benthamiana.

[0175]N. benthamiana transformations were based upon the leaf discmethod of Horsch et al. (1985) and Horsch and Klee (1986). Leavesapproximately 90 mm wide were removed from young plants approximately 10cm in height and surface-sterilised by immersion in 2% bleach for 15minutes, followed by one rinse in 70% ethanol and five 10-minute washesin sterile water. Discs of 0.5 cm diameter were punched from the leavesusing a flame-sterilised size 3 cork borer incubated on pre-callusingplates (Horsch et al. (1985)) in continuous light for 24 hours at 22° C.The leaf discs were then dipped in an overnight LB culture ofAgrobacterium tumefaciens strain GV3101 (O.D. 600=0.1) containing the B6cosmid, transferred to fresh pre-callusing plates, and returned to thegrowth chamber. After 48 hours the discs were transferred to selectionmedia, containing phospinothricin (PPT) at 5 mg per litre, andcarbenicillin at 500 mg per litre to kill the Agrobacterium. Transformedexplants produced green shoots after 3-5 weeks that were excised using aflame-sterilised scalpel and transferred to Magenta pots containingrooting media (Horsch et al. (1985). Upon rooting, shoots were grown inmoistened, sterilised soil comprising John Innes No.3 compost, coarsegrit, peat and vermiculite. Pots were placed in glass jars and coveredwith transparent film for 3-4 days to retain high humidity. Plants werethen maintained at 23° C. under short day conditions to delay flowering.

[0176] Twelve transgenic Nicotiana benthamiana plants (T1-T12) wereregenerated from a screen of 28 leaf disc explants.

[0177] Single leaves from the putative transgenic tobacco plants weresprayed with three applications of 30 mg per litre BASTA herbicidecontaining glufosinate ammonium were sprayed over 6 days. All wereresistant to BASTA, confirming that they had been transformed.

[0178] Results of N. benthamiana Transformed with B6

[0179] The transgenic plants T5 and T6 and plants transformed withvector only, as control, were inoculated with E. cichoracearum UCSC1. T5and T6 plants were resistant to E. cichoracearum, whereas the controlswere susceptible, and the fungus grew as a superficial white myceliumclearly visible to the naked eye.

[0180]N. benthamiana Transformation with SE7.5

[0181] The SE7.5 construct was made as follows: A 7.5 kb SmaI and EcoRIfragment starting 1637 bp upstream of RPW8.2 (SmaI site of B6 cosmidclone in the SLJ755I5 vector obtained from the JIC) and ending 2912 bpdownstream of RPW8.1 (EcoRI site inside the ATPK41A gene) was obtainedby SmaI complete digestion of first and then partial EcoRI digestion ofthe B6 cosmid clone. The 7.5 kb fragment was recovered, purified, andligated to SamI-EcoRI digested pBIN19-Plus binary vector (obtained fromJIC). The resulting plasmid carryied AtRPW8.1 and AtRPW8.2 genomicsequence including their native promoters and was named SE7.5 (see FIG.3). It was introduced to E coli (DH10B from GIBCOL-BRL).

[0182] Agrobacterium (strain GV3101, obtained from The Sainsbury Lab,JIC) was used for transformation. The Agrobacterium strain was grown for48 hours at 30° C. in 10 ml LB medium supplemented with 25 μg/mlRifampicin, 25 μg/ml Gentamycin, 50 μg/ml Kanamycin. About 100 μl ofthis cell culture was then added to 10 ml fresh LB medium withoutantibiotics, and shaken for further 24 hours at 30° C. The Agrobacteriumwas then diluted with liquid MS medium to achieve an OD₆₀₀ of 0.1.

[0183] Tobacco leaves from young plants were surface-sterilized byimmersion in 2% bleach (12% active Cl⁻w/v) followed by one rinse in 70%ethanol and five 10 minute-washes in sterile water. Discs of 0.5 cm.diameter were punched from the leaves using a flame-sterilized size 3cork borer. The leaf discs were incubated on regeneration plates, sealedwith micropore tape and kept under continuous light for 24 hours at 22°C. in a growth cabinet. The leaf discs were then dipped in the dilutedAgrobacterium, swirling occasionally. Excess liquid was removed withfilter paper and leaf discs were transferred to fresh regenerationmedia, sealed and returned to the growth cabinet. After two daysco-cultivation at 22° C., the tobacco leaf discs were transferred toselective regeneration medium containing 500 mg/L Carbenicillin and 100mg/L Kanamycin as selective agents (about 10 leaf discs per 9cm-diameter petri dish) and cultured at 22° C. under continuous light.Transformed explants produced green shoots after 3-5 weeks which wereexcised and placed on rooting medium containing 200 mg/L Carbenicillin,and 100 mg/L Kanamycin in sealed glass jars (Magenta pots). Rootingplants were transferred and grown in moistened, sterilized soil. Plantswere maintained in a sealed propagation tray to retain high humidityunder short day condition for a number of days, then transferred tonormal humidity conditions in the glasshouse.

[0184] About 4 weeks old transgenic T1 plants and wild type plants wereinoculated with Erysiphe cichoracearum UCSC1, and their phenotypes werechecked 10 days after inoculation. Erysiphe cichoracearum UCSC1 wasobtained from the Carnegie Institute, Washington, Stanford USA, where itwas originally identified on Arabidopsis Col-0 plants grown in theirgreenhouse, and was subsequently purified from a single colony.

[0185] Results of N. benthamiana Transformation with SE7.5

[0186] More than 20 T1 lines of transgenic N. benthamiana weregenerated. The presence of AtRPW8 genes was confirmed by PCR usingAtRPW8.1-specific primers (5′- ATGCCGATTGGTGAGCTTGCGATA-3′ and areverse, 5′-TCAAGCTCTTATTTTACTACAAGC-31). and AtRPW8.2-specific primers(5′-ATGATTGCTGAGGTTGCCGCA-3′ and 5′-TCAAGAATCATCACTGCAGAACGT-3′).

[0187] T2 progenies of 5 T1 lines were tested with UCSC1 isolate, whichis the only isolate we found that can mildly infect N. benthamiana. Allthe 5 lines showed no or very little fungal growth (disease rating: 0,or 0-1) and some T2 plants developed obvious necrotic lesions (HR),whereas, the wild type plants supported more fungal growth andsporulation (disease rating:1, or 1-2), and had no obvious necroticlesions.

[0188]Nicotiana tabacum Transformation with SE7.5

[0189]N. tabacum variety Petit Gerard was used for transformation. Thetransformation procedures were the same as that used for N. benthamiana,except that axenic tobacco leaves were used as explants and A.tumefaciens strain LBA4404 containing SE7.5 construct was used fortransformation.

[0190] Results of Nicotiana tabacum Transformation with SE7.5

[0191] Eighteen T1 lines carrying both AtRPW8.1 and AtRPW8.2 genes weregenerated. And 4 of them were tested with Erysiphe orontii MGH(originally identified and purified on Arabidopsis plants by Dr. FredAusubel's group in Massachusetts General Hospital, Harvard University)along with the wild type. The wild type N. tobaccum plants were fullysusceptible to this isolate (disease rating 2-3 or 3), while three T1plants were completely resistant (no visible fungus; disease rating 0)and surprisingly, had no visible necrotic lesions. One T1 plantsupported a little fungal growth (disease rating 0-1˜1) and had somenecrotic lesions.

[0192] Transfer of RPW8 to Wheat, Barley and to Tomato.

[0193] Those skilled in the art are well aware of methods for theproduction of stable, fertile transgenic plants of Triticum aestivum(wheat), Hordeum sativum (barley), and Lycopersicon esculentum (tomato)by Agrobacterium-mediated transformation and transfer of RPW8.1 andRPW8.2 to wheat, barley, and tomato can be achieved using any preferredmethods. Transgenic plants so produced can be tested for resistance topowdery mildew pathogens that affect the relevant crop species.

[0194] Generally speaking, RPW8.1 and RPW8.2 may be amplified by theprimers specified such as GACCCGTACAGTACTAAGTCTA andGATTTCCGAAATTGATTACAAGAA (for RPW8.1) and AACTCTTCACCTCGAGAGCTAACA andAGTCGTTTGACACAATTGGGACAT (for RPW8.2) and cloned into a binary vector,introduced into the specified strain of Agrobacterium tumefaciens byelectroporation, and used to transform appropriate tissue from thedifferent plant species.

[0195] Transformation of Tomato with RPW8.1 and RPW8.2:

[0196] One suitable method employs A. tumefaciens strain LBA4404 withthe binary vector of Filliati (1987). Tomato seeds are germinated understerile conditions, and cotyledon explants are placed on filter paper ontobacco cell feeder cultures and co-cultivated with A. tumefaciens asspecified in Filliati (1987) and McCormick (1986). Selection is appliedwith kanamycin (McCormick, 1987), and shoots that develop aretransferred to rooting medium, and then to soil. Tests for the transgene(McDonnell, 1987) are used to confirm transgenic plants. These are grownon to collect seed. Progeny from these primary transgenic plants arethen tested for resistance to powdery mildew.

[0197] For example, Lycopersicon esculentum transformation with SE7.5may be carried out as follows:

[0198] Preparation of tomato seedlings: Tomato variety Moneymaker wasused for transformation. tomato seeds were treated with 70% ethanol for2 minutes and rinsed once with sterile water. Then, the seeds wereimmersed in 10% Domestos and shaken for 3 hours followed by 4 times ofwashes with water. The seeds were left in water and shaken at 25° C.overnight. About 25 seeds were put into tubs containing germinationmedium and left in 4° C. for 2 weeks. Seedlings were grown undercontinuous light at 22° C. growth cabinet for 7-10 days.

[0199] Preparation of Agrobacterium culture: A. tumefaciens strainLBA4404 containing SE7.5 construct was used for transformation. Thestrain was inoculated to 10 ml LB containing 25 μg/ml Rifampicin, 25μg/ml Gentamycin, 50 μg/ml Kanamycin and the cultured in a 28° C. shakerfor 28 hours.

[0200] Setting up feeder layers: 1 ml of fine tobacco suspension culturewas spread evenly onto plates containing MS medium with 0.5 mg/L 2,4-D,0.6% agar. The plates were left unsealed and stacked, and put undercontinuous light at 22° C. growth cabinet for 24 hours.

[0201] Incubation of explants: A piece of Whatman no.1 filter paper wasplaced on top of the feeder plate and wet completely. Any air bubbleswere excluded. Young and still expanding cotyledons of tomato seedlingsprior to true leaf formation were used as explants. Cotyledon tips werecut off and two more transverse cuts were made to give two explants ofabout 0.5 cm. long. The explants were transferred to a new petri dishfull of water to prevent any damage during further cutting. Once anumber of explants were collected in the pool, they were dabbed ontosterile filter paper and 30-40 explants were then placed onto a feederplate, with topside down. Petri dishes were placed unsealed and stackedunder continuous light at 22° C. growth cabinet for 8 hours.

[0202] Co-cultivation: Agrobacterium cells were spun down andresuspended in MS medium containing 3% sucrose to an OD₅₉₀ of 0.4-0.5.The explants from feeder plates were completely immersed in bacterialsuspension and then removed and dabbed on filter paper before returnedto the original feeder plate. The explants were co-cultivated with theagrobacterial cells under the same conditions as used in thepre-incubation phase for 40 hours.

[0203] Selection: The explants were taken from the feeder layers and puton tomato regeneration plates containing 500 mg/L Carbenicillin, and 100mg/L Kanamycin for selection. Cotyledons explants were placed on mediumwith the right side upwards ensuring good contact with the nutrients anddrugs. About 10 explants were placed in every plate and the plates werereturned to the growth cabinet. The explants were transferred to freshmedium every 2-3 weeks. Once the regenerating material became too largefor petri dishes, it was then put into larger pot (Magenta vessel).

[0204] Plant regeneration: Regenerated shoots were cut from the explantsand put onto rooting medium containing 200 mg/L Carbenicillin, 100 mg/LKanamycin. Once the shoots developed roots, they were removed the mediumby washing the root gently under running water and then transferred tohydrated, autoclaved Jiffy pots (containing peat) and placed inside asealed propagation tray to maintain humidity in short day growth room.Once roots were seen growing through the Jiffy pots, the putativetransgenic plants were transferred to bigger pots containing soil andkept in the glasshouse. Confirmation of transgene: DNA was extractedfrom regenerated T1 tomato plants and used for PCR amplification withAtRPW8.1 and AtRPW8.2 specific primers.

[0205] Pathogen test: About 4 weeks old T1 tomato plants were inoculatedwith Oidium lycopersici Oxford and examined forresistance/susceptibility 10 DPI.

[0206] Transformation of barley with RPW8.1 and RPW8.2

[0207] Barley is routinely transformed by Agrobacterium tumefaciens(Tingay et al. 1997), and this method may be used as described for theproduction of plants transgenic for RPW8.1 and RPW8.2.

[0208]A. tumefaciens carrying RPW8.1 and RPW8.2 in a binary vector undercontrol of a promoter constitutively expressed in barley, and with thebar gene as selectable marker on the T-DNA, is co-cultivated withimmature barley embryo explants. Selection is made forbialaphos-resistant cultures, from which plants are regenerated usingstandard methods (Tingay et al. 1997). From more than 1,500 embryos, itis generally possible to recover more than 50 plants, more than 10 ofwhich will grow to maturity and be fertile. Tests on their progeny forthe marker gene (bar gene conferring bialaphos resistance) will identifyindividuals with the transgene at a single locus, which are then used totest for resistance to the powdery mildew pathogen.

[0209] Transformation of Wheat with RPW8.1 and RPW8.2

[0210] A rapid Agrobacterium tumefaciens-mediated transformation systemis used for wheat (Duncan et al. 1997). This uses either freshlyisolated immature embryos, precultured immature embryos, or embryogeniccalli as explants. The explants are inoculated with a disarmed A.tumefaciens strain C58 (ABI) harboring the binary vector pMON18365containing RPW8.1 and RPW8.2 under control of a promoter constitutivelyexpressed in wheat, and a selectable marker, the neomycinphosphotransferase II gene. The inoculated immature embryos orembryogenic calli are selected on G418-containing media. Transgenicplants are regenerated from the three types of explants. The procedureis rapid, and the total time required from inoculation to theestablishment of plants in soil is generally 2.5 to 3 months, with mostor all transformants morphologically normal, having the insert stablyintegrated and segregating in a Mendelian fashion. T2 plants are testedfor resistance to the wheat powdery mildew pathogen.

Example 9 The RPW8 Promoters

[0211] As shown in Example 8, the SE7.5 construct containing AtRPW8.1and AtRPW8.2 under their corresponding promoters demonstrates that theseAtRPW8 promoters work in tobacco (N. benthamiana and N. tobaccum).

[0212] In a separate experiment, the same construct (SE7.5) causes celldeath when transiently expressed in N. bentamiana by agro-infiltration.

[0213] The RPW8 Promoter is also Wound Activated

[0214] The following were cloned into binary vector pBI101 in front ofthe GUS translation start by using HindIII and XbaI restriction sites:1000 bp sequence upstream of AtRPW8.1 translation start, 1000 bpsequence upstream of AtRPW8.2 translation start, and 496 bp sequenceupstream of AtHR3 translation start. All the three fusion constructswere introduced into Arabidopsis Col-0 via Agrobacterium-mediatedtransformation. T1 transgenic plants were selected on MS platescontaining 50 mg/L Kanamycin.

[0215] Mature leaves of at least 10 T1 plants from each construct werewounded by fine forceps and then immediately immersed in GUS stainingsolution (50 mM Na₃PO₄, pH7.0, 1.0 mM X-Glucuronide), and incubated for˜14 hours. Two week-old T 2 seedlings selected on MS plates containing50 mg/L Kanamycin were treated with SA and JA (2.5 ml of 1 mM SA and 2.5ml of 0.4 mM JA were added to the small petri dishes (4.5 cM indiameter) containing the plants) for 72 hours. Seedling were thentransferred into GUS staining solution for ˜14 hours.

[0216] Initial results indicated that wounding induced GUS activity inmost of the T1 transgenic plants carrying either one the 3 promoter-GUSfusion constructs. SA seemed to induce GUS activity in their T2 plants,while JA did not. These observations suggest that AtRPW8 promoters arewounding and SA responsive.

Example 10 Over-Expression of RPW8 Induces Cell Death

[0217] Many Arabidopsis T1 lines carrying AtRPW8.1 and AtRPW8.2 genomicsequence (either construct SE14, EE7.5 or EE6.2) showed necrotic lesionson leaves in the absence of powdery mildew pathogens. In order tofurther investigate whether the cell death on these plants arespontaneous, we generated T4 lines homozygous for the transgene from oneT1 line, named SE14-24, which shows the most severe cell deathphenotype.

[0218] Southern analysis indicated this line had a single insertion,however, that insertion might have had multiple copies of AtRPW8tandemly linked, as it was indicated by the higher intensity of thetransgene band of SE14-24 (not shown). Quantitative RT-PCR confirmedthat SE14-24 T4 plants have much higher level of AtRPW8.1 and AtRPW8.2mRNA (data not shown).

[0219] SE14-24 T4 plants growing in sterile MS medium normally do notdevelop necrotic lesions, but they do have spontaneous cell death whentransferred to sterile soil or perlite. High light and low humiditypromote cell death , while, high temperature (30° C.), high humidity anddark/low light suppress/alleviate cell death phenotype. It was alsoconfirmed that the spontaneous cell death in the SE14-24 line startsfrom the palisade mesophyll cells and the cell death is associated withlocalised H202 accumulation.

[0220] General Methods Used (Except Where Stated Otherwise)

[0221] Pathology

[0222]O. lypcopersicum Oxford and E. orontii MGH were propagated onLycopersicon esculentum cv Moneymaker; E. chichoracearum UCSC1 waspropagated on squash, and E. cruciferarum UEA1 on oilseed rape(8). A.thaliana plants were inoculated according to Xiao et al. (1997)(8).

[0223] Molecular Biology

[0224] General Methods Were According to Sambrook et al. 1989(19)

[0225] Molecular Markers

[0226] Selected YAC from the AtEM1 contig on chromosome 3(http://genome-www3.stanford.edu/atdb_welcome.html) and BAC ends wereisolated(20), sequenced and used to develop CAPS and RFLP markerspolymorphic between Ms-0 and Ler for the fine-mapping of RPW8. CAPSmarkers included: X1-6 from cosmid X1-11 end (primersATCCGCCTCTTTCTTTTGGTTTTC and GTGTTACTTTTCTACAGCCAGAG; polymorphismrevealed with BstNI,); B9 from cosmid B6 end (primersGTCTGAATCCGTCAAGCCTTCG and TCCATGCTTCTATATTGAAGAGC, polymorphismrevealed with CfoI), and 6I2-L from BAC 6I2 end (primersGATTGTATAGGTTGGTTGATGAG and GCATCTCATTGACCTCCCTATC, polymorphismrevealed with HindIII). RFLP markers included : 8E1-R from YAC 8E1(probe amplified with primers CAGCTTCCTTCACCGTCTCATGG andCCAGGAAAATAACGGTGACGATC; polymorphism revealed with CfoI); and 3B3-Lfrom BAC 3B3 end (probe amplified with primers GTCATCATCTAAAGAGGATAAGGand GGTTGAAAAAGTGGCTTTGGATG, polymorphism revealed with NsiI). RFLPmarker Atpk41A was an EST (L05561; probe amplified with primersATGGATCCGGCGACTAATTCACC and TGTCCTCAGGAATCTCAGAGAGC; polymorphismrevealed with CfoI).

[0227] Cosmid Library

[0228] Genomic DNA from accession Ms-0 was partially digested withSau3AI and fractions 15-25 kb were ligated into the BamHI site of vectorSLJ755I5, packaged into lambda using Gigapack□ III XL Packaging Extractkit (Stratagene), and propagated in ˜60,000 colony forming units of E.coli strain DH1OB (GIBCO-BRL).

[0229] DNA Sequencing

[0230] Overlapping EcoRI and HindIII fragments of cosmids B6 and J4-2were ligated into appropriate sites in pBluescript II SK⁺ (Stratagene),cloned in E. coli strain XL-Blue (Statagene), and sequenced. RPW8alleles from A. thaliana accessions were amplified by PCR from genomicDNA with primers specific for RPW8.1 (GACCCGTACAGTACTAAGTCTA andGATTTCCGAAATTGATTACAAGAA) and for RPW8.2 (AACTCTTCACCTCGAGAGCTAACA andAGTCGTTTGACACAATTGGGACAT). Products from 4 independent PCRs were pooledand sequenced. DNA sequences were assembled with the Staden DNA analysispackage and analysed with programmes at HGMP (hgmp.mrc.ac.uk website).

[0231] Transcript Analysis

[0232] 3′RACE and 5′RACE were according to the manufacturer'sinstruction (GIBCO BRL). Gene specific primers for RPW8.1 were: 3′RACE:AATGGACACTAAACTTGCTGAAGT and 5′RACE: CCACAACTATTATGCTTCT, and is nestedprimer GAACCAAAAACGGCTCGATACTAA. Gene-specific primers for RPW8.2 were3′RACE: GCTAAATTACGATGGGTGGTAGAT and nested primerCGATGGGTGGTAGATGTGGATGTT, and 5′RACE: GGATCGCACGGTTTGT and nested primerCTGAACTTCTTGCGTACGTTTCT. PCR products were cloned into pGEM-T easyvector (Promega) in E. coli strain XL-Blue, and sequenced.

[0233]A. thaliana Transformations

[0234] Restriction sites detected in the sequence of cosmid B6 were usedto make sub-clones in vector SLJ755I5 propagated in E. coli strainDH1OB. RPW8.1 and RPW8.2 cDNAs were amplified by RT-PCR using Pfu-Turbo(Stratagene) with primers for RPW8.1 (CCGGAATTCATGCCGATTGGTGAGCTTGCGATAand CGCGGATCCTCAAGCTCTTATTTTACTACAAGC) and RPW8.2(CCGGAATTCATGATTGCTGAGGTTGCCGCA and CCGGGATCCTCAAGAATCATCACTGCAGAACGT),and cloned into the EcoRI-BamHI site of pKMB(21) for expression undercontrol of the constitutive viral 35 S promoter in A. thaliana Col-0.Clones were maintained in E. coli DH1OB. Agrobacterium tumefaciensstrain GV3101 was transformed with plasmids by electroporation, and usedfor stable transformation of A. thaliana accession Col-0(10).

[0235] Misc Materials

[0236] We thank F. M. Ausubel for E. orontii MGH, M. Bardin for E.cichoracearum isolates, J. R. Botella for pKMB, S. Covey for cauliflowermosaic virus infections, S. Gurr for O. lypcopersicum Oxford, J. Jonesfor pSLJ755I5, J. Parker for P. parasitica Noco2, Ohio Stock Centre forBAC filters containing IGF and TAMU libraries, K. Schrick for CAPSmarker g19397, M. Stammers for YAC clones and for BAC filters, and X.Dong for A. thaliana Col-0 transgenic for NahG.

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[0258] Additional References:

[0259] Brettell R, 1997. Plant Journal Agrobacteriumtumefaciens-mediated barley transformation 11: 1369-1376

[0260] Duncan D R, Conner T W, Wan Y C, 1997. Plant Physiology, 115:971-980

[0261] Filliati, J. J, et al. Bio/Technology (1987) 5:726-730

[0262] Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D.,Rogers, S. G. and I Fraley, R. T. (1985) A simple general method fortransferring genes into plants. Science 227,1229-1231.

[0263] Horsch R. B. and Klee, H. J .(1986) Rapid assay of foreign geneexpression in leaf discs transformed by Agrobacterium tumefaciens; Roleof the T-DNA borders in the transfer process. Proc. Natl. Acad. Sci. USA83,4428-4432.

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[0265] McDonnell et al. 1987. Molecular Plant Biology Reporter5:380-386.

[0266] Tingay S, McElroy D, Kalla R, Fieg S, Wang M B, Thornton S,

[0267] Sequence Listing 2. The RPW8 Locus mRNA RPW8.1 complement 13878 .. . 14719 CDS complement(join(13990-14155;14353-14633)) /label = RPW8.1,powdery mildew resistance gene /note = “Transcription start: 14719;                       exon1: 14719-14353;                       Intron:14352-14156;                        Exon2: 14155-13878;           Transcription end: 13878;             Protein sequence:mpigelaigavlgvgagaiydrfrkardisfvhrlcatilsiepflvqidkrskvegsplrevnerltcflelayvfveaypklrrrqvlrkyryikaietielalrsiivvdfqvdqwddikeikakisemdtklaevisacskira” mRNA RPW8.2 complement 15904 . . .16829 CDS complement(join(16015-16243;16372-16667)) /label = RPFW8.2,powdery mildew resistance gene /note = “Transcription start: 16829;                       exon1: 16829-16372;                       Intron:16371-16244;                        Exon2: 16243-15904           Transcription end: 15904;             Protein sequence:miaevaaggalglalsvlheavkrakdrsvttrfilhrleatidsitpivvqidkfseemedstsrkvnkrlklllenavslveenaelrrrnvrkkfrymrdikefeaklrwvvdvdvqvnqladikelkakmseistkldkimpqpkfeihigwcsgktnrairftfcsdds” nucleotidesequence: 13801 catgaaacat agatctcaaa agaagcgaaa taaaaagatt attgttaattattattttga 13861 taaaattaca catagattga gaaagagttt ttcaataatt atggqgaataagagagagag 13921 agagagaaat agatttccga aattgattac aagaagaaat aatttcaacaaagtctctgt 13981 ttttttttat caagctctta ttttactaca agcagaaata acttcagcaagtttagtgtc 14041 catttcagat atcttggcct tgatttcttt gatatcgtcc cattgatcaacttgaaaatc 14101 cacaactatt atgcttctta atgcaagttc tatcgtttcg attgctttgatgtacctaaa 14161 gataaacaga acaaacataa tactcgtgtt atttttccac aacatgataggttttgtacg 14221 tttagtgttt ggagattatc gaaatcatgt aaaaaaaatt gttacaaagaagaagatatt 14281 tttctctaaa ccattaaact aagaaattag gcgatccaaa aaccaatagaaattcatgtc 14341 atatatacga acctgtactt cctgagtact tgtctgcgtc tgagtttcggataagcctca 14401 acaaaaacat aagctaattc aaggaaacac gtgagacgtt cgttgacttcccttaatggt 14461 gaaccttcca ctttactccg cttatcgatt tgaaccaaaa acggctcgatactaaggatt 14521 gtagcgcaga gacggtgtac gaaagatata tctcttgctt ttctgaaccggtcgtaaatg 14581 gcttgggctc caactccaag aacagcccct atcgcaagct caccaatcggcattttttga 14641 aagtagttgt ttagctctcg aggtgaatat agaggaatct atgtacatggaaggatggaa 14701 ccatattaaa tagttttatg tttaacaagt taacgagtgg ttttaattatatgaagacaa 14761 ttcaagagat tgactcatag acttagtact gtacgggtca acaactctctctttttctag 14821 gtaagaggag atcgttggat ctatatgcaa gttgtcgtga gtattaaattacgtagaata 14881 ttattgaatt acgtcgaaga agcgagagtc aatctcactc tcaatggttaacttgtacat 14941 ttagaagaag gaaaaatcaa cgaagttggc tgagtaagaa gtgaagaagaaaaacagtga 15001 agaaagccaa aaagcagaag aggaaaatgg tggtatcaac taaaaatatttcaacaaagg 15061 aagttactac taaaaatatt tcaacaaaag aagttactac taaaaataaatactttgcat 15121 gttgcagtat atatttaaaa tttagaaata attatatcta ttaaaaaatcattttgtaac 15181 agatgttcga ttatgatata tagaattatt ttgtagacgt tttataaaatagtttaaaaa 15241 attatattga agatatgaga tgaaccacaa tacgtatttt tatttttcgtattttcaaat 15301 aaactcttat tattatatga aatctgaatt agcccagaat attattagatttggtttata 15361 atttaatctc aaaattttct tccaaactga aaacagaaaa aaaaaaaaaaaaaaaaagaa 15421 gaagaagaag aagaagttaa aaaccactaa tctgaaagat ccactctaatttgtataaat 15481 ttttcgtttt aagttcaaag atgggatcaa atcaaatgag aagaatccttaaaaactttc 15541 atctttatgt aagaagcaaa agcaaattta gttaagcttt tttctaagttctttatatct 15601 tctttcagca ttaattcatt atccacaact ttgttatact cattatccttcaaacttgat 15661 tgtattgagt ttgcttctcc gttgatccta atacgctaag ttcaactctttgtaacaact 15721 ttgttcttta aagcattttg agttctaaat aaacaaattg agagaccaatgtggcagata 15781 atcgtcattt tgagatcgtt tgttgttttt tactctacaa actttggattcacatacata 15841 tatatatata tatatataga tatatatata tatatatatt gtaatgtaatgtatagtata 15901 tttctgaatt tctctttgtt taataaccat tggcacattt atttattttcaaagtatgtc 15961 attagattat tcatattaat acatatatat gagtcgtttg acacaattgggacatcaaga 16021 atcatcactg cagaacgtaa atcggatcgc acggtttgtt tttcctgaacaccagccgat 16081 gtggatttca aacttcggtt gaggcattat tttgtcaagt ttagtgctgatttcagacat 16141 cttggccttg agttctttga tatcagccaa ttgattaact tgaacatccacatctaccac 16201 ccatcgtaat ttagcttcga actctttgat atctctcatg tacctaaagataaacaacac 16261 aaatataata cacatgttat tgacttaatt catagtaaat gttaggttttgatagattta 16321 gtactgttgg gagtttatgg aaatcacata taggaactat ttagcacaaacctgaacttc 16381 ttgcgtacgt ttctgcgtct cagctccgca ttctcctcaa caagagaaacagcgttctca 16441 aggagaagct taagacgttt attgactttc ctcgatgttg aatcttccatttcttcactg 16501 aacttatcaa tttgaaccac caacggtgtg atactatcga ttgtagcttcgagacggtgt 16561 aagatgaatc ttgtggttac agatctatct tttgctcttt tgacggcctcgtggaggaca 16621 ctgagagcaa gtccaagagc accccctgcg gcaacctcag caatcattttcttgaaatta 16681 gtttgttagc tctcgaggtg aagagttttt gatgagttat attgatgatattattttgtt 16741 tggtaagaaa aatataagac catctattat attatataga ggtgaatatttataattcct 16801 ttttcttctc aaatatttgg taaagtgttg ctctattaat tcacataatgttagtattat 16861 acacaaatat tataagggtg aatgcaatga gaaatctatg aacatggaagtcttttgctt 16921 aacaattaag ccgtgtagtt tgtataaagt caaacggatg ttctttgtttccgtaacttc 16981 ctacgaaaga gtgtgaataa gagatgtgtg gaccgcttgg taaagtaccatgcagttaga 17041 agcatgtacg gggtagtgaa acgtcgattt ttattataaa ataaaataataaacgatatg 17101 tgttggaggc gtatatatat taataaatag ttaaataaca aaattaaatcgtcttttact 17161 ttttttatag ctaataaaat caaatagttt aaagtcaatt ttagatcattgtcagtaaaa 17221 acatcattaa actcaagtct ttcaaagtta atttaattaa atttatgcagaaaattcata 17281 aaacatagat ctcaaaagaa gcaaaataaa aagattattg ttaattattattttgataaa 17341 attacacata gattgagaaa gagtttttca atcattattg ggaagaagggaggaagaaaa 17401 gaaaaaacag atttctgaaa ttgattataa gaagaaataa tttcaacagtctctgttttt 17461 ttaaatcaag ttcttatttt attacaaagt gaaataattt cagatatcttggccttgatt 17521 tctttggtat ctttctaaaa aacaaattta gagaccaatg tggcagagaatcgtcatttt 17581 gggatcgttt gttgtttttt actctacaaa ctttggattc acatacatattatatgtatt 17641 gtaatgtaat gagtaatata tttctgaatg tctctttgtt tacgttacattggcacattt 17701 atgaagacaa aagacgtttt tgattaatta tattgatgat atatataaagacaaaagacg 17761 tttcacaaaa tattaaaacc ttaggaaaga caccccattt atcatcaatggaggtgctct 17821 tagataacaa tctagaatcc ttatcgcttt agacagctgt gttattgactagtcatcatc 17881 taaagaggat aaggattgga aacgatttga attggagacc aagtgcttggagagtaagct 17941 tagggttgtc tttgtatgtg tgtatatata ctcctcaaga tcgatcaataacatcaagca 18001 ctttttcaac cattcttagt ctttacaatt aatgtacgaa gaggattattatttattaaa 18061 ttacgaaaaa gaagtgaaaa tcgatctaaa tgattgactt tttacgtagaatcgtcgaat 18121 tgcatgtaca tttccaccga aattccaaaa atctgaatta caaataagttggaaccgatc 18181 gatcttgttt tgtatattta cgtacaaggc agacgtacat acatgtagtttggattatca 18241 tatgtatgat caacgcaatt ttcgtgaata gaaacgtgaa tactaacaatttcggtgaat 18301 acctaccgta aatactaaca ttaaaatcta tgacttctta aaataataatcaatcaaact 18361 tttacatttg attttatatt ttcctcagtt tttaggccta tgatacacctgccttctcaa 18421 aatattagtt ccgtgatgtt tgctccatct aaggtggata tcgatc

[0268] Sequence Listing 3: The cDNA Nucleotide Sequence of RPW8.1 fromMs-0 is Aligned with that of RPW8.1 Homologues Isolated by PCR fromother A. thaliana Accessions.1                                                   50 RPW8.1c-MsATGCCGATTG GTGAGCTTGC GATAGGGGCT GTTCTTGGAG TTGGAGCCCA RPW8.1c-WaATGCCGATTG GTGAGCTTGC GATAGGGGCT GTTCTTGGAG TTGGAGCCCA RPW8.1c-KasATGCCGATTG GTGAGCTTGC GATAGGGGCT GTTCTTGGAG TTGGAGCCCA RPW8.1c-G24ATGCCGATTG GTGAGCTTGC GATAGGGGCT GTTCTTGGAG TTGGAGCCCA RPW8.1c-CanATGCCGATTG GTGAGCTTGC GATAGGGGCT GTTCTTGGAG TTGGAGCCCA RPW8.1c-NdATGCCGATTG GTGAGCTTGC GATAGGGGCT GTTCTTGGAG TTGGAGCCCA RPW8.1c-SyATGCCGATTG GTGAGCTTGC GATAGGGGCT GTTCTTGGAG TTGGAGCCCA RPW8.1c-WsATGCCGATTG GTGAGCTTGC GATAGGGGCT GTTCTTGGAG TTGGAGCCCA RPWB.1c-LerATGCCGATTG GTGAGCTTGC GATAGGGGCT GTTCTTGGAG TTGGAGCCCA51                                                 100 RPW8.1c-MsAGCCATTTAC GACCGGTTCA GAAAAGCAAG AGATATATCT TTCGTACACC RPW8.1c-WaAGCCATTTAC GACCGGTTCA GAAAAGCAAG AGATATATCT TTCGTACACC RPW8.1c-KasAGCCATTTAC GACCGGTTCA GAAAAGCAAG AGATATATCT TTCGTACACC RPW8.1c-C24AGCCATTTAC GACCGCTTCA GAAAAGCAAG AGATATATCT TTCGTACACC RPW8.1c-CanAGCCATTTAC GACCGCTTCA GAAAAGCAAG AGATATATCT TTCGTACACC RPW8.1c-NdAGCCATTTAC GACCGCTTCA GAAAAGCAAG AGATATATCT TTCGTACACC RPW8.1c-SyAGCCATTTAC GACCGGTTCA GAAAAGCAAG AGATATATCT TTCGTACACC RPW8.1c-WsAGCCATTTAC GACCGCTTCA GAAAAGCAAG AGATATATCT GTCGTAAACC RPW8.1c-LerAGCCATTTAC GACCGCTTCA GAAAAGCAAG AGATATATCT GTCGTAAACC101                                                150 RPW8.1c-MsGTCTCTGCGC TACAATCCTT AGTATCGAGC CGTTTTTGGT TCAAATCGAT RPW8.1c-WaGTCTCTGCGC TACAATCCTT AGTATCGAGC CGTTTTTGGT TCAAATCGAT RPW8.1c-KasGTCTCTGCGC TACAATCCTT AGTATCGAGC CGTTTTTGGT TCAAATCGAT RPW8.1c-C24GTCTCTGCGC TACAATCCTT AGTATCGAGC CGTTTTTGGT TCAAATCGAT RPW8.1c-CanGTCTCTGCGC TACAATCATT AGTATCGAGC CGTTTTTGGT TCAAATCGAT RPW8.1c-NdGTCTCTGCGC TACAATCATT AGTATCGAGC CGTTTTTGGT TCAAATCGAT RPW8.1c-SyGTCTCTGCGC TACAATCCTT AGTATCGAGC CGTTGTTGGT TCAAATCGAT RPW8.1c-WsGTCTCTGCGC TACAATCATT AGTATCAGGC CGTTGTTGGT TCAAATCGAT RPW8.1c-LerGTCTCTGCGC TACAATCATT AGTATCAGGC CGTTGTTGGT TCAAATCGAT151                                                200 RPW8.1c-MsAAGCGGAGTA AAGTGGAAGG TTCACCATTA AGGGAAGTCA ACGAACGTCT RPW8.1c-WaAAGCGGAGTA AAGTGGAAGG TTCACCATTA AGGGAAGTCA ACGAACGTCT RPW8.1c-KasAAGCGGAGTA AAGTGGAAGG TTCACCATTA AGGGAAGTCA ACGAACGTCT RPW8.1c-C24AAGCGGAGTA AAGTGGAAGG TTCACCATTA AGGGAAGTCA ACGAACGTCT RPW8.1c-CanAAGCGGAGTA AAGTGGAAGG TTCACCATTA AGGGAAGTTA ACGAACGTCT RPW8.1c-NdAAGCGGAGTA AAGTGGAAGG TTCACCATTA AGGGAAGTTA ACGAACGTCT RPW8.1c-SyAAGCGGAGTA AAGTGGAAGG TTCACCATTA AGGGAAGTCA ACGAACGTCT RPW8.1c-WsAAGCGGAGTA AAGTGGAAGG TTCACCATTA ACGGAAGTCA ACGAACGTCT RPW8.1c-LerAAGCGGAGTA AAGTGGAAGG TTCACCATTA AGGGAAGTCA ACGAACGTCT201                                                250 RPW8.1c-MsCACGTGTTTC CTTGAATTAG CTTATGTTTT TGTTGAGGCT TATCCGAAAC RPW8.1c-WaCACGTGTTTC CTTGAATTAG CTTATGTTTT TGTTGAGGCT TATCCGAAAC RPW8.1c-KasCACGTGTTTC CTTGAATTAG CTTATGTTTT TGTTGAGGCT TATCCGAAAC RPW8.1c-C24CACGTGTTTC CTTGAATTAG CTTATGTTTT TGTTGAGGCT TATCCGAAAC RPW8.1c-CanCACGTGTTTC CTTGAATTAG CTTATGTTTT AGTTGAGGCT TATCCGAAAC RPW8.1c-NdCACGTGTTTC CTTGAATTAG CTTATGTTTT AGTTGAGGCT TATCCGAAAC RPW8.1c-SyCACGTGTTTC CTTGAATTAG CTTATGTTTT AGTTGAGGCT TATCCGAAAC RPW8.1c-WsCACGTGTTTC CTTGAATTAG CTTATGTTTT AGTTGAGGCT TATCCGAAAC RPW8.1c-LerCACGTGTTTC CTTGAATTAG CTTATGTTTT TGTTGAGGCT TATCCGAAAC251                                                300 RPW8.1c-NsTCAGACGCAG ACAAGTACTC AGGAAGTACA GGTACATCAA AGCAATCGAA RPW8.1c-WaTCAGACGCAG ACAAGTACTC AGGAAGTACA GGTACATCAA AGCAATCGAA RPW8.1c-KasTCAGACGCAG ACAAGTACTC AGGAAGTACA GGTACATCAA AGCAATCGAA RPW8.1c-C24TCAGACGCAG ACAAGTACTC AGGAAGTACA GGTACATCAA AGCAATCGAA RPW8.1c-CanTCAGACGCAG ACAAGTACTC AGGAAGTACA GGTGCATCAA AGCAATCGAA RPW8.1c-NdTCAGACGCAG ACAAGTACTC AGGAAGTACA GGTGCATCAA AGCAATCGAA RPW8.1c-SyTCAGACGCAG ACAAGTACTC AGGAAGTACA GGTACATCAA AGCAATCGAA RPW8.1c-WsTCAGACGCAG ACAAGTACTC AGGAAGTACA GGTACATCAA AGCAATCGAA RPW8.1c-LerTCAGAGGCAG ACAAGTACTC AGGAAGTACA GGTACATCAA AGCAATCGAA301                                                350 RPW8.1c-MsACGATAGAAC TTGCATTAAG AAGCATAATA GTTGTGGATT TTCAAGTTGA RPW8.1c-WaACGATAGAAC TTGCATTAAG AAGCATAATA GTTGTGGATT TTCAAGTTGA RPW8.1c-KasACGATAGAAC TTGCATTAAG AAGCATAATA GTTGTGGATT TTCAAGTTGA RPW8.1c-C24ACGATAGAAC TTGCATTAAG AAGCATAATA GTTGTGGATT TTCAAGTTGA RPW8.1c-CanACGATAGAAC TTGCATTAAG AAGGATAATA GTTGTGGATT TTCAAGTTGA RPW8.1c-NdACGATAGAAC TTGCATTAAG AAGGATAATA GTTGTGGATT TTCAAGTTGA RPW8.1c-SyACGATAGAAC TTGCATTAAG AAGCATAATA GTTGTGGATT TTCAAGTTGA RPW8.1c-WsACGATAGAAC TTGCATTAAG AAGCATAATA GTTGTGGATT TTCAAGTTGA RPW8.1c-LerACGATAGAAC TTGCATTAAG AAGCATAATA GTTGTGGATT TTCAAGTTGA351                                                400 RPW8.1c-MsTCAATGGGAC GAT....... .......... .......... .......... RPW8.1c-WaTCAATGGGAC GAT....... .......... .......... .......... RPW8.1c-KasTCAATGGGAC GAT....... .......... .......... .......... RPW8.1c-C24TCAATGGGAC GAT....... .......... .......... .......... RPW8.1c-CanTCAATGGGAC GATATCAAAG AAATCAAGGC CAAGATATCT GAAACGGACA RPW8.1c-NdTCAATGGGAC GATATCAAAG AAATCAAGGC CAAGATATCT GAAACGGACA RPW8.1c-SyTCAATGGGAC GAT....... .......... .......... .......... RPW8.1c-WsTCAATGGGAC GAT....... .......... .......... .......... RPW8.1c-LerTCAATGGGAC GAT....... .......... .......... ..........401                                                450 RPW8.1c-Ms.......... .......... ......ATCA AAGAAATCAA GGCCAAGATA RPW8.1c-Wa.......... .......... ......ATCA AAGAAATCAA GGCCAAGATA RPW8.1c-Kas.......... .......... ......ATCA AAGAAATCAA GGCCAAGATA RPW8.1c-C24.......... .......... ......ATCA AAGAAATCAA GGCCAAGATA RPW8.1c-CanCTAAACTTGC TGATCAATGG GACGATATCA AAGAAATCAA GGCCAAGATA RPW8.1c-NdCTAAAGTTGC TGATCAATGG GACGATATCA AAGAAATCAA GGCCAAGATA RPW8.1c-Sy.......... .......... ......ATCA AAGAAATCAA GGCCAAGATA RPW8.1c-Ws.......... .......... ......ATCA AAGAAATCAA GGCCAAGATA RPW8.1c-Ler.......... .......... ......ATCA AAGAAATCAA GGCCAAGATA451                                                500 RPW8.1c-MsTCTGAAATGG ACACTAAACT TGCTGAAGTT ATTTCTGCTT GTAGTAAAAT RPW8.1c-WaTCTGAAATGG ACACTAAACT TGCTGAAGTT ATTTCTGCTT GTAGTAAAAT RPW8.1c-KasTCTGAAATGG ACACTAAACT TGCTGAAGTT ATTTCTGCTT GTAGTAAAAT RPW8.1c-C24TCTGAAATGG ACACTAAACT TGCTGAAGTT ATTTCTGCTT GTAGTAAAAT RPW8.1c-CanTCTGAAATGG ACACTAAACT TGCTGAAGTT ATTTCTGCTT GTAGTAAAAT RPW8.1c-NdTCTGAAATGG ACACTAAACT TGCTGAAGTT ATTTCTGCTT GTAGTAAAAT RPW8.1c-SyTCTGAAATGG ACACTAAACT TGCTGAAGTT ATTTCTGCTT GTAGTAAAAT RPW8.1c-WsTCTGAAATGG ACACTAAACT TGCTGAAGTT ATTTCTGCTT GTAGTAAAAT RPW8.1c-LerTCTGAAATGG AGACTAAACT TGCTGAAGTT ATTTCTGCTT GTAGTAAAAT 501 RPW8.1c-MsAAGAGCTTGA RPW8.1c-Wa AAGAGCTTGA RPW8.1c-Kas AAGAGCTTGA RPW8.1c-C24AAGAGCTTGA RPW8.1c-Can AAGAACTTGA RPW8.1c-Nd AAGAACTTGA RPW8.1c-SyAAGAGCTTGA RPW8.1c-Ws AAGAGCTTGA RPW8.1c-Ler AAGAACTTGA1                                                   50 RPW8.1p-MsMPIGELAIGA VLGVGAQAIY DRFRKARDIS FVHRLCATIL SIEPFLVQID RPW8.1p-Wa---------- ---------- ---------- ---------- ---------- RPW8.1p-Kas---------- ---------- ---------- ---------- ---------- RPW8.1p-C24---------- ---------- ---------- ---------- ---------- RPW8.1p-Can---------- ---------- ---------- ---------I ---------- RPW8.1p-Nd---------- ---------- ---------- ---------I ---------- RPW8.1p-Sy---------- ---------- ---------- ---------- ----L----- RPW8.1p-Ws---------- ---------- ---------- V-N------I --R-L----- RPW8.1p-Ler---------- ---------- ---------- V-N------I --R-L-----51                                                 100 RPW8.1p-MsKRSKVEGSPL REVNERLTCF LELAYVFVEA YPKLRRRQVL RKYRYIKAIE RPW8.1p-Wa---------- ---------- ---------- ---------- ---------- RPW8.1p-Kas---------- ---------- ---------- ---------- ---------- RPW8.1p-C24---------- ---------- ---------- ---------- ---------- RPW8.1p-Can---------- ---------- ------L--- ---------- ----C----- RPW8.1p-Nd---------- ---------- ------L--- ---------- ----C----- RPW8.1p-Sy---------- ---------- ------L--- ---------- ---------- RPW8.1p-Ws---------- ---------- ------L--- ---------- ---------- RPW8.1p-Ler---------- ---------- ---------- ---------- ----------101                                                150 RPW8.1p-MsTIELALRSII VVDFQVDQWD .......... .......... .DIKEIKAKI RPW8.1p-Wa---------- ---------- .......... .......... .--------- RPW8.1p-Kas---------- ---------- .......... .......... .--------- RPW8.1p-C24---------- ---------- .......... .......... .--------- RPW8.1p-Can-------R-- ---------- DIKEIKAKIS ETDTKLADQW D--------- RPW8.1p-Nd-------R-- ---------- DIKEIKAKIS ETDTKLADQW D--------- RPW8.1p-Sy---------- ---------- .......... .......... .--------- RPW8.1p-Ws---------- ---------- .......... .......... .--------- RPW8.1p-Ler---------- ---------- .......... .......... .---------151                169 RPW8.1p-Ms SEMDTKLAEV ISACSKIRA RPW8.1p-Wa---------- --------- RPW8.1p-Kas ---------- --------- RPW8.1p-C24---------- --------- RPW8.1p-Can ---------- --------T RPW8.1p-Nd---------- --------T RPW8.1p-Sy ---------- --------- RPW8 1p-Ws---------- --------- RPW8.1p-Ler ---------- --------T

[0269] Sequence Listing 4: The Predicted Amino Acid Sequence of RPW8.1from Ms-0 is Aligned with RPW8.1 Homologues Isolated by PCR from otherA. thaliana Accessions1                                                   50 RPW8.1p-MsMPIGELAIGA VLGVGAQAIY DRFRKARDIS FVHRLCATIL SIEPFLVQID RPW8.1p-Wa---------- ---------- ---------- ---------- ---------- RPW8.1p-Kas---------- ---------- ---------- ---------- ---------- RPW8.1p-C24---------- ---------- ---------- ---------- ---------- RPW8.1p-Can---------- ---------- ---------- ---------I ---------- RPW8.1p-Nd---------- ---------- ---------- ---------I ---------- RPW8.1p-Sy---------- ---------- ---------- ---------- ----L----- RPW8.1p-Ws---------- ---------- ---------- V-N------I --R-L----- RPW8.1p-Ler---------- ---------- ---------- V-N------I --R-L-----51                                                 100 RPW8.1p-MsKRSKVEGSPL REVNERLTCF LELAYVFVEA YPKLRRRQVL RKYRYIKAIE RPW8.1p-Wa---------- ---------- ---------- ---------- ---------- RPW8.1p-Kas---------- ---------- ---------- ---------- ---------- RPW8.1p-C24---------- ---------- ---------- ---------- ---------- RPW8.1p-Can---------- ---------- ------L--- ---------- ----C----- RPW8.1p-Nd---------- ---------- ------L--- ---------- ----C----- RPW8.1p-Sy---------- ---------- ------L--- ---------- ---------- RPW8.1p-Ws---------- ---------- ------L--- ---------- ---------- RPW8.1p-Ler---------- ---------- ------L--- ---------- ----------101                                                150 RPW8.1p-MsTIELALRSII VVDFQVDQWD .......... .......... .DIKEIKAKI RPW8.1p-Wa---------- ---------- .......... .......... .--------- RPW8.1p-Kas---------- ---------- .......... .......... .--------- RPW8.1p-C24---------- ---------- .......... .......... .--------- RPW8.1p-Can-------R-- ---------- DIKEIKAKIS ETDTKLADQW D--------- RPW8.1p-Nd-------R-- ---------- DIKEIKAKIS ETDTKLADQW D--------- RPW8.1p-Sy---------- ---------- .......... .......... .--------- RPW8.1p-Ws---------- ---------- .......... .......... .--------- RPW8.1p-Ler---------- ---------- .......... .......... .---------151                169 RPW8.1p-Ms SEMDTKLAEV ISACSKIRA RPW8.1p-Wa---------- --------- RPW8.1p-Kas ---------- --------- RPW8.1p-C24---------- --------- RPW8.1p-Can ---------- --------T RPW8.1p-Nd---------- --------T RPW8.1p-Sy ---------- --------- RPW8.1p-Ws---------- --------- RPW8.1p-Ler ---------- --------T

[0270] Sequence Listing 5: The cDNA Nucleotide Sequence of RPW8.2 fromMs-0 is Aligned with that of RPW8.2 Homologues Isolated by PCR fromother A. thaliana Accessions.1                                                   50 RPW8.2c-MsATGATTGCTG AGGTTGCCGC AGGGGGTGCT CTTGGACTTG CTCTCAGTGT RPW8.2c-WaATGATTGCTG AGGTTGCCGC AGGGGGTGCT CTTGGACTTG CTCTCAGTGT RPW8.2c-KasATGATTGCTG AGGTTGCCGC AGGGGGTGCT CTTGGACTTG CTCTCAGTGT RPW8.2c-C24ATGATTGCTG AGGTTGCGGC AGGGGGTGCT CTTGGACTTG CTCTCAGTGT RPW8.2c-CanATGATTGCTG AGGTTGCCGC AGGGGGTGCT CTTGGACTTG CTCTCAGTGT RPW8.2c-NdATGATTGCTG AGGTTGCGGC AGGGGGTGCT CTTGGACTTG GTCTCAGTGT RPW8.2c-SyATGATTGCTG AGGTTGCCGC AGGGGGTGGT CTTGGACTTG CTCTCAGTTT RPW8.2c-WsATGATTGCTG AGGTTGCGGC AGGGGGTGCT CTTGGACTTG CTCTCAGTTT RPW8.2c-LerATGATTGCTG AGGTTGCGGC AGGGGGTGCT CTTGGACTTG CTCTCAGTGT51                                                 100 RPW8.2c-MsCCTCCACGAG GCCGTCAAAA GAGCAAAAGA TAGATCTGTA ACCACAAGAT RPW8.2c-WaCCTCCACGAG GCCGTCAAAA GAGCAAAAGA TAGATCTGTA ACCACAAGAT RPW8.2c-KasCCTCCACGAG GCCGTCAAAA GAGCAAAAGA TAGATCTGTA ACCACAAGAT RPW8.2c-C24CCTTCAAGAG GCCGTCAAAA GAGCAAAAGA TAGATCTGTA ACCACAAGAT RPW8.2c-CanCCTCCACGAG GCCGTCAAAA GAGCAAAAGA TAGATCTGTA ACCACAAGAT RPW8.2c-NdCCTCCACGAG GCCGTCAAAA GAGCAAAAGA TAGATCTGTA ACCACAAGAT RPW8.2c-SyCCTCCACGAG GCCGTCAAAA GAGCAAAAGA TAGATCTGTA ACCACAAGAT RPW8.2c-WsCCTCCACGAG GCCGTCAAAA GAGCAAAAGA TAGATCTGTA ACCACAAGAT RPW8.2c-LerCCTCCACGAG GCCGTCAAAA GAGCAAAAGA TAGATCTGTA ACCACAAGAT101                                                150 RPW8.2c-MsTCATCTTACA CCGTCTCGAA GCTACAATCG ATAGTATCAC ACCGTTGGTG RPW8.2c-WaTCATCTTACA CCGTCTCGAA GCTACAATCG ATAGTATCAC ACCGTTGGTG RPW8.2c-KasTCATCTTACA CCGTCTCGAA GCTACAATCG ATAGTATCAC ACCGTTGGTG RPW8.2c-C24TCATCTTACA CCGTCTCGAA GCTACAATCG ATAGTATCAC TCCGTTGGTG RPW8.2c-CanTCATCTTACA CCGTCTCGAA GCTACAATCG ATAGTATCAC ACCGTTGGTG RPW8.2c-NdTCATCTTACA CCGTCTCGAA GCTACAATCG ATAGTATCAC TCCGTTGGTG RPW8.2c-SyTCATCTTACA CCGTCTCGAA GCTACAATCG ATAGTATCAC TCCGTTGGTG RPWB.2c-WsTCATCTTACA CCGTCTCGAA GCTACAATCG ATAGTATCAC TCCGTTGGTG RPW8.2c-LerTCATCTTACA CCGTCTCGAA GCTACAATCG ATAGTATCAC TCCGTTGGTG151                                                200 RPW8.2c-MsGTTCAAATTG ATAAGTTCAG TGAAGAAATG GAAGATTCAA CATCGAGGAA RPW8.2c-WaGTTCAAATTG ATAAGTTCAG TGAAGAAATG GAAGATTCAA CATCGAGGAA RPW8.2c-KasGTTCAAATTG ATAAGTTCAG TGAAGAAATG GAAGATTCAA CATCGAGGAA RPW8.2c-C24GTTCAAATTG ATAAGTTCAG TGAAGAAATG GAAGATTCAT CATCGAGGAA RPW8.2c-CanGTTCAAATTG ATAAGTTCAG TGAAGAAATG GAAGATTCAT CATCGAGGAA RPW8.2c-NdGTTCAAATTG ATAAGTTCAG TGAAGAAATG GAAGATTCAT CATCGAGGAA RPW8.2c-SyGTTCAAATTG ATAAGTTCAG TGAAGAAATG GAAGATTCAT GATCGAGGAA RPWS.2c-WsGTTCAAATTG ATAAGTTCAG TGAAGAAATG GAAGATTCAT CATCGAGGAA RPW8.2c-LerGTTCAAATTG ATAAGTTCAG TGAAGAAATG GAAGATTCAT CATCGAGGAA201                                                250 RPW8.2c-MsAGTCAATAAA CGTCTTAAGC TTCTCCTTGA GAACGCTGTT TCTCTTGTTG RPWB.2c-WaAGTCAATAAA CGTCTTAAGC TTCTCCTTGA GAACGCTGTT TCTCTTGTTG RPWB.2c-KasAGTCAATAAA CGTCTTAAGC TTCTCCTTGA GAACGCTGTT TCTCTTGTTG RPW8.2c-C24AGTCAATAAA CGTCTTAAGC TTCTCCTTGA GAAGGCTGTT TCTCTTGTTG RPW8.2c-CanAGTCAATAAA CGTCTTAAGC TTCTCCTTGA GAACGCTGTT TCTCTTGTTG RPW8.2c-NdAGTCAATAAA CGTCTTAAGC TTCTCCTTGA GAAGGCTGTT TCTCTTGTTG RPWB.2c-SyAGTCAATAAA CGTCTTAAGC TTCTCCTTGA GAACGCTGTT TGTCTTGTTG RPW8.2c-WsAGTCAATAAA CGTCTTAAGC TTCTCCTTGA GAACGCTGTT TCTCTTGTTG RPW8.2c-LerAGTCAATGAA CGTCTTAAGC TTCTCCTTGA GAACGCTGTT TCTCTTGTTG251                                                300 RPW8.2c-MsAGGAGAATGC GGAGCTGAGA CGCAGAAACG TACGCAAGAA GTTCAGGTAC RPW8.2c-WaAGGAGAATGC GGAGCTGAGA CGCAGAAACG TACGCAAGAA GTTCAGGTAC RPW8.2c-KasAGGAGAATGC GGAGCTGAGA CGCAGAAACG TACGCAAGAA GTTCAGGTAC RPW8.2c-C24AGGAGAATGC GGAGCTGAGA CGCAGAAACG TACGCAAGAA GTTCAGGTAC RPW8.2c-CanAGGAGAATGC GGAGCTGAGA CGCAGAAACG TACGCAAGAA GTTCAGGTAC RPW8.2c-NdAGGAGAATGC GGAGCTGAGA CGCAGAAACG TAGGCAAGAA GTTCAGGTAC RPW8.2c-SyAGGAGAATGC GGAGCTGAGA CGCAGAAACG TACGCAAGAA GTTCAGGTAC RPW8.2c-WsAGGAGAATGC GGAGCTGAGA CGCAGAAACG TACGCAAGAA GTTCAGGTAC RPW8.2c-LerAGGAGAATGC GGAGCTGAGA CGCAGAAACG TACGCAAGAA GTTCAGGTAC301                                                350 RPW8.2c-MsATGAGAGATA TCAAAGAGTT CGAAGCTAAA TTACGATGGG TGGTAGATGT RPW8.2c-WaATGAGAGATA TCAAAGAGTT CGAAGCTAAA TTACGATGGG TGGTAGATGT RPW8.2c-KasATGAGAGATA TCAAAGAGTT CGAAGCTAAA TTACGATGGG TGGTAGATGT RPW8.2c-C24ATGAGAGATA TCAAAGAGTT CGAAGCTAAG ATACGATGGG TGGTAGGTGT RPW8.2c-CanATGAGAGATA TCAAAGAGTT CGAAGCTAAA TTACGATGGG TGGTAGGTGT RPW8.2c-NdATGAGAGATA TCAAAGAGTT CGAAGCTAAA TTACGATGGG TGGTAGGTGT RPW8 2c-SyATGAGAGATA TCAAAGAGTT CGAAGCTAAA TTACGATGGG TGGTAGGTGT RPW8.2c-WsATGAGAGATA TCAAAGAGTT CGAAGCTAAA TTACGATGGG TGGTAGGTGT RPW8.2c-LerATGAGAGATA TCAAAGAGTT GGAAGCTAAA TTACGATGGG TGGTAGGTGT351                                                400 RPW8.2c-MsGGATGTTCAA GTTAATCAAT TGGCTGATAT CAAAGAACTC AAGGCCAAGA RPW8.2c-WaGGATGTTCAA GTTAATCAAT TGGCTGATAT CAAAGAACTC AAGGCCAAGA RPW8.2c-KasGGATGTTCAA GTTAATCAAT TGGCTGATAT CAAAGAACTC AAGGCCAAGA RPW8.2c-C24GGATGTTCAA GTTAATCAAT TGGCTGATAT CAAAGAACTC AAGGCCAAGA RPW8.2c-CanGGATGTTCAA GTTAATCAAT TGGCTGATAT CAAAGAACTC AAGGCCAAGA RPW8.2c-NdGGATGTTCAA GTTAATCAAT TGGCTGATAT CAAAGAACTC AAGGCCAAGA RPW8.2c-SyGGATGTTCAA GTTAATCAAT TGGCTGATAT CAAAGAACTC AAGGCCAAGA RPW8.2c-WsGGATGTTCAA GTTAATCAAT TGGCTGATAT CAAAGAACTC AAGGCCAAGA RPW8.2c-LerGGATGTTCAA GTTAATCAAT TGGCTGATAT CAAAGAACTC AAGGCCAAGA401                                                450 RPW8.2c-MsTGTCTGAAAT CAGCACTAAA CTTGACAAAA TAATGCCTCA ACCGAAGTTT RPW8.2c-WaTGTCTGAAAT CAGCACTAAA CTTGACAAAA TAATGCCTCA ACCGAAGTTT RPW8.2c-KasTGTCTGAAAT CAGCACTAAA CTTGACAAAA TAATGCCTCA ACCGAAGTTT RPW8.2c-C24TGTCTGAAAT CAGCACTAAA CTTGACAAAA TAATGCCTCA ACCGAAGTTT RPW8.2c-CanTGTCTGAAAT CAGCACTAAA CTTGACAAAA TAATGCCTCA ACCGAAGTTT RPW8.2c-NdTGTCTGAAAT CAGCACTAAA CTTGACAAAA TAATGCCTCA AGCGAAGTTT RPW8.2c-SyTGTCTGAAAT CAGCACTAAA CTTGACAAA. TAATGCCTCA ACCGAAGTTT RPW8.2c-WsTGTCTGAAAT CAGCACTAAA CTTGACAAA. TAATGCCTCA ACCGAAGTTT RPW8.2c-LerTGTCTGAAAT CAGCACTAAA CTTGACAAAA TAATGCCTCA ACCGAAGTTT451                                                500 RPW8.2c-MsGAAATCCACA TCGGCTGGTG TTCAGGAAAA ACAAACCGTG CGATCCGATT RPW8.2c-WaGAAATCCACA TCGGCTGGTG TTCAGGAAAA ACAAACCGTG CGATCCGATT RPW8.2c-KasGAAATCCACA TCGGCTGGTG TTCAGGAAAA ACAAACCGTG CGATCCGATT RPW8.2c-C24GAAATCCACA TCGGCTGGTG TTCAGGAAAA AAAAACCGTG CGATCCGATT RPW8.2c-CanGAAATCCACA TCGGCTGGTG TTCAGGAAAA ACAAACCGTG CGATCCGATT RPW8.2c-NdGAAATCCACA TCGGCTGGTG TTCAGGAAAA AAAAAGCGTG CGATCCGATT RPW8.2c-SyGAAATCCACA TCGGCTGGTG TTCAGGAAAA AAAAACCGTG CGATCCGATT RPW8.2c-WsGAAATCCACA TCGGCTGGTG TTCAGGAAAA AAAAACCGTG CGATCCGATT RPW8.2c-LerGAAATCCACA TCGGCTGGTG TTCAGGAAAA ACAAACCGTG CGATCCGATT501                                                525 RPW8.2c-MsTACGTTCTGC AGTGATGATT CTTGA RPW8.2c-Wa TACGTTCTGC AGTGATGATT CTTGARPW8.2c-Kas TACGTTCTGC AGTGATGATT CTTGA RPW8.2c-C24 TACGTTCTGCAGTGATGATT CTTGA RPW8.2c-Can TACGTTCTGC AGTGATGATT CTTGA RPW8.2c-NdTACGTTCTGC AGTGATGATT CTTGA RPW8.2c-Sy TACGTTCTGC AGTGATGATT CTTGARPW8.2c-Ws TACGTTCTGC AGTGATGATT CTTGA RPW8.2c-Ler TACGTTCTGC AGTGATGATTCTTGA

[0271] Sequence Listing 6: The Predicted Amino Acid Sequence of RPW8.2from Ms-0 is Aligned with RPW8.2 Homologues Isolated by PCR from otherA. thaliana Accessions1                                                   50 RPWB.2p-MsMIAEVAAGGA LGLALSVLHE AVKRAKDRSV TTRFILHRLE ATIDSITPLV RPW8.2p-Wa---------- ---------- ---------- ---------- ---------- RPW8.2p-Kas---------- ---------- ---------- ---------- ---------- RPW8.2p-C24---------- --------Q- ---------- ---------- ---------- RPW8.2p-Can---------- ---------- ---------- ---------- ---------- RPW8.2p-Nd---------- ---------- ---------- ---------- ---------- RPW8.2p-Sy---------- ------F--- ---------- ---------- ---------- RPW8.2p-Ws---------- ------F--- ---------- ---------- ---------- RPW8.2p-Ler---------- ---------- ---------- ---------- ----------51                                                 100 RPW8.2p-MsVQIDKFSEEM EDSTSRKVNK RLKLLLENAV SLVEENAELR RRNVRKKFRY RPW8.2p-Wa---------- ---------- ---------- ---------- ---------- RPW8.2p-Kas---------- ---------- ---------- ---------- ---------- RPW8.2p-C24---------- ---S------ ---------- ---------- ---------- RPW8.2p-Can---------- ---S------ ---------- ---------- ---------- RPW8.2p-Nd---------- ---S------ ---------- ---------- ---------- RPW8.2p-Sy---------- ---S------ ---------- ---------- ---------- RPW8.2p-Ws---------- ---S-----E ---------- ---------- ---------- RPW8.2p-Ler---------- ---S-----E ---------- ---------- ----------101                                                150 RPW8.2p-MsMRDIKEFEAK LRWVVDVDVQ VNQLADIKEL KAKMSEISTK LDKIMPQPKF RPW8.2p-Wa---------- ---------- ---------- ---------- ---------- RPW8.2p-Kas---------- ---------- ---------- ---------- ---------- RPW8.2p-C24---------- I----G---- ---------- ---------- ---------- RPW8.2p-Can---------- -----G---- ---------- ---------- ---------- RPW8.2p-Nd---------- -----G---- ---------- ---------- ---------- RPW8.2p-Sy---------- -----G---- ---------- ---------- ---------- RPW8.2p-Ws---------- -----G---- ---------- ---------- ---------- RPW8.2p-Ler---------- -----G---- ---------- ---------- ----------151                    174 RPW8.2p-Ms EIHIGWCSGK TNRAIRFTFC SDDSRPW8.2p-Wa ---------- ---------- ---- RPW8.2p-Kas ---------- -------------- RPW8.2p-C24 ---------- K--------- ---- RPW8.2p-Can -------------------- ---- RPW8.2p-Nd ---------- K--------- ---- RPW8.2p-Sy.......... .......... .... RPW8.2p-Ws .......... .......... ....RPW8.2p-Le ---------- ---------- ----

[0272] Sequence Listing 7: BrHR1 Genomic Sequence (756 bp)Atgcctattggtgaagttattgtaggggctgctcttggaattactctgcaagtgcttcatgaagctatcataaaagcaaaagatagatcttcaaccaaaaaaagtatcttggaccgcctcgatgctacaatctccaggatcactccgttggtggttcatgtcgataagatcagcaaaagagtagaagattctgagaggaaagtcattgaagaactcaagcgtcttcttgaaaaggctgtttctcttgttgaggcttatgcagaactcagacgcagaaacctacacaagaagcataggtttgtatagtttatataatacatgaaatacttgaaaaagtctttgtgatttcttaaaatgtttttatttggtttacataatatttatgtgttgttgatatataggtgcaagagtagaatcaaagagttagaagtttcattaagatggatgatagatgtggatgttcaagtcaaccaatggctagatatcaaaaaactcgtggttaagatgtctgaaatgaacacaaaactcgacaagatcacgtgccaaccaactgatggtagttgtttcaagagcaatgatagcacatcaccagtgttttcacaaagtagtagtagtctcgaagcaacagacggatcttcagaggaagatgaagaagaaagcccaagtaatggatctgaaccaaggatcgatatccacctgcgatggagttcaagaaaaggaagaaaagatcgtgagatccgattcatggccaagtga

[0273] Sequence Listing 8: Predicted BrHR1 cDNA Sequence (651 bp)Atgcctattggtgaagttattgtaggggctgctcttggaattactctgcaagtgcttcatgaagctatcataaaagcaaaagatagatcttcaaccaaaaaaagtatcttggaccgcctcgatgctacaatctccaggatcactccgttggtggttcatgtcgataagatcagcaaaagagtagaagattctgagaggaaagtcattgaagaactcaagcgtcttcttgaaaaggctgtttctcttgttgaggcttatgcagaactcagacgcagaaacctacacaagaagcataggtgcaagagtagaatcaaagagttagaagtttcattaagatggatgatagatgtggatgttcaagtcaaccaatggctagatatcaaaaaactcgtggttaagatgtctgaaatgaacacaaaactcgacaagatcacgtgccaaccaactgatggtagttgtttcaagagcaatgatagcacatcaccagtgttttcacaaagtagtagtagtctcgaagcaacagacggatcttcagaggaagatgaagaagaaagcccaagtaatggatctgaaccaaggatcgatatccacctgcgatggagttcaagaaaaggaagaaaagatcgtgagatccgattcatggccaagtga

[0274] Sequence Listing 9: Predicted BrHR1 Protein Sequence (217 aa)Mpigevivgaalgitlqvlheaiikakdrsstkksildrldatisritplvvhvdkiskrvedserkvieelkrllekavslveayaelrrrnlhkkhrcksrikelevslrwmidvdvqvnqwldikklvvkmsemntkldkitcqptdgscfksndstspvfsqssssleatdgsseedeeespsngsepridihlrwssrkgrkdreirfmak

[0275] Sequence Listing 10: BrHR2 Genomic Sequence (753 bp)Atgcctattggtgaggttattgtaggggctgctcttggaattactctgcaagtgcttcatcaagctatcataaaagcaaaagatagatcttcaaccacaaaatgtatcttggtccgcctcgatgctacaatctccaggatcactccgttggtggttcatgtcgataagatcagcaaaagagtagaagattctgagaggaaagtcattgaagaactcaagcgtcttcttgaaaaggctgtttctcttgttgaggcttatgcagaactcagacgcagaaacctacacaagaagcattggtttgtatagtttatataatacatgaaatacttgaaaaagtctttgtgatttcttaaaatgtttttatttggtttacataatatttatgtgttgttgatatataggtacaagagtagaatcaaagagttagaagcttcattaagatggatggtagatgtggatgttcaagtcaaccaatggctagatatcaaagaactcgtggctaagatgtctgaaatgaacacaaaactcgacaagatcacgagccaaccaactgatggtagttgtttcaagagcaatgatagcatatcaccagtgttatcacaaagtagtaggatcgaagcaacagacggatcttcagaggaagatgaagaagaaagctcaagtaatggatccgaaccaaggatcgatatccacctgcgatggagttcaagaaaaggaagaaaagatcgtgagatccgattcacggccaagtga

[0276] Sequence Listing 11: Predicted BrHR2 cDNA Sequence (648 bp)Atgcctattggtgaggttattgtaggggctgctcttggaattactctgcaagtgcttcatcaagctatcataaaagcaaaagatagatcttcaaccacaaaatgtatcttggtccgcctcgatgctacaatctccaggatcactccgttggtggttcatgtcgataagatcagcaaaagagtagaagattctgagaggaaagtcattgaagaactcaagcgtcttcttgaaaaggctgtttctcttgttgaggcttatgcagaactcagacgcagaaacctacacaagaagtataggtacaagagtagaatcaaagagttagaagcttcattaagatggatggtagatgtggatgttcaagtcaaccaatggctagatatcaaagaactcgtggctaagatgtctgaaatgaacacaaaactcgacaagatcacgagccaaccaactgatggtagttgtttcaagagcaatgatagcatatcaccagtgttatcacaaagtagtaggatcgaagcaacagacggatcttcagaggaagatgaagaagaaagctcaagtaatggatccgaaccaaggatcgatatccacctgcgatggagttcaagaaaaggaagaaaagatcgtgagatccgattcacggccaagtga

[0277] Sequence Listing 12: Predicted BrHR2 Protein Sequence (216 aa)Mpigevivgaalgitlqvlhqaiikakdrssttkcilvrldatisritplvvhvdkiskrvedserkvieelkrllekavslveayaelrrrnlhkkyryksrikeleaslrwmvdvdvqvnqwldikelvakmsemntkldkitsqptdgscfksndsispvlsqssrieatdgsseedeeesssngsepridihlrwssrkgrkdreirftak

[0278] Sequence Listing 13: BrHR3 Genomic Sequence (746 bp)Atgccgattggtgaggttcttgtaggggctgctcttggaattacactccaagtgcttcatgaagccatcataaaagcaaaacatagatctttaaccacaaaatgtatcttggaccgcctcgatgctacaatctccaggatcactccgttggtggttcatgtcgataagatcagcaaaggggtagaagattctcagaggaaagtcattgaagacctcaagcgtcttcttgaaaaggctgtttttcttgttgaggcttatgcagaactcagacgcagaaacctactcaagaagtttaggtatgtatagtttatatagtacatgaaatgcttgaaaagtctttgtgattcttaaaatgtttttgttttgtttatataatatatatgtgtgtgttgttgatatctaggtacaagagtagaatcaaagagttggaagcttctttaagatggatggtagaggtggatgttcaagtcaaccaatggttggatatcaaacaactcctggccaagatgtttgaaatgaacactaaactcgagaggatcacgtgcccaccaactgattgtaattgtttcaagagaaatgatagcacatcaccagtgatatcacaaagtagtaatcaaaatatactcgaagcaacagacggatcgtcagaggaagacgaagaagaaagcccaaggattgatatccaccttcgatggagttcaagaaaaggagctaaagatcgtgagatccgattcatggtcaagtga

[0279] Sequence Listing 14: Predicted BrHR3 cDNA Sequence (639 bp)Atgccgattggtgaggttcttgtaggggctgctcttggaattacactccaagtgcttcatgaagccatcataaaagcaaaacatagatctttaaccacaaaatgtatcttggaccgcctcgatgctacaatctccaggatcactccgttggtggttcatgtcgataagatcagcaaaggggtagaagattctcagaggaaagtcattgaagacctcaagcgtcttcttgaaaaggctgtttttcttgttgaggcttatgcagaactcagacgcagaaacctactcaagaagtttaggtacaagagtagaatcaaagagttggaagcttctttaagatggatggtagaggtggatgttcaagtcaaccaatggttggatatcaaacaactcctggccaagatgtttgaaatgaacactaaactcgagaggatcacgtgcccaccaactgattgtaattgtttcaagagaaatgatagcacatcaccagtgatatcacaaagtagtaatcaaaatatactcgaagcaacagacggatcgtcagaggaagacgaagaagaaagcccaaggattgatatccaccttcgatggagttcaagaaaaggagctaaagatcgtgagatccgattcatggtcaagtga

[0280] Sequence Listing 15: Predicted BrHR3 Protein Sequence (213 aa)mpigevlvgaalgitlqvlheaiikakhrslttkcildrldatisritplvvhvdkiskgvedsqrkviedlkrllekavflveayaelrrrnllkkfryksrikeleaslrwmvevdvqvnqwldikqllakmfemntkleritcpptdcncfkrndstspvisqssnqnileatdgsseedeeespridihlrwssrkgakdreirfmvk

[0281]

1 75 1 27689 DNA Arabidopsis thaliana 1 gaattcggtg aaattttgcc aaatttatactatagactat agagtgtgaa ttattttatg 60 tacttttggt atgaagataa gaattttggtattatcacaa tcatcatgaa aataaaggct 120 aactaacttc attgattata gttgtttatttggataagtt atttttggga ggagaaatta 180 attggaacaa tttaatagac attgacagggtattgtatta ttatacatat acagatgcat 240 ttataatcct gccaactctt gaatgtgcattagatttcat ttcaggtcat cttttttttt 300 gtttggtaag gccactaata ttaagttaaccaatcataat ttcatattat ggattttaat 360 tggaatctag cgtttttttt ttttttccatttgttagact atttactttt ctttataatt 420 tttttttgtg atttttattc aagcgaagtagggttttcaa tttttaattc tgtattttac 480 attgttctag gacataaaac atcgcaacaaaatagaagtt gttattttca gcagtttcta 540 acgagttcaa attttattta gttgatagtttgataccggt ttcttatcaa ttacaaatct 600 ataaccaatg caaaaaattt tatatggaaatgtaaacgat gacactaatt aaaaccaata 660 aattatcggg accctaaatt ataagtttcaagttattctg ctgatatcgt ttaaaaaagc 720 aaacttaaat ggtatggaca ctctcaggtccgacggtatt aatattgcct tataacatta 780 tggtccatat aataatatac atgattaccccactattaac aatagataac aacaaattta 840 tcaattgcga tctctgaaga ctataactttgatattcgta ttaacttgta tatacacaga 900 acagaagcaa taaaacttgg aacatgtcacagaaattcaa actattttgt tctctactat 960 tggttttagg tttcaagaga gaagtatagagaaaatgtga tgtaataata atcggagatc 1020 gacgtaagat gttcgtggtg ggtaaaggtgggacccataa aagagtctgg tggtccagct 1080 agataaataa ttaatactta ctaataaattaaataatgga gaaataaata gtgtaaatag 1140 taaagaagta ggtttgttcc ttccttaattaacgctattg ccatttggat ttgtggtggg 1200 gacgactctc tttcgatgct tcctcgacaaataaagctgt gccttctcta tgttcacttc 1260 aagggtaaaa acgtcattat atcactattttttttttttt tttcaagttc cactacgaca 1320 cgtcagtaat tcaattcgtt aatggttattggtcaaacga cgttaacttc tctcacgtga 1380 gattaaacac aacaaataac tgctacttgcaaggttttga caatttaaaa caatcttagc 1440 ctacgttaca ggcgcaaaag attcattcaaatgtttaatt tgaggtttag tgtaattaat 1500 ttaatgtaaa ctaaaagcaa tacaacgtgagactgaatga aactaatgat tatgttgtat 1560 ttttataatt cgtatgatga tgaatatgaaacttgttaaa ttgttgcttg ttccgtccga 1620 cacattgaga agccaattca aaatttgcagctagctatca ttttttaccc acttatatat 1680 tcgtattatt gatacttcaa atgcatgcacacgtatagtg gtaaacatat tcattatgta 1740 tccctctcta taagattttt atgtgttgatgaaattttta tgttccctat tttattgtaa 1800 tcagtttgaa tatattttcg tttttttgaatagctcatca aaccgaaacc cttcacatga 1860 tcacctaaaa atagtaaatt ttcaagatcatgcattcgaa caccattgat aagagaaaac 1920 tcattggttg tcaaaatact atctataaatccatatttaa tatttcaatg ttagaggttt 1980 cagtttcagt ttgtgactgt gttattaactatcatctata tacgataaat gcagaaaaat 2040 aatgtttgca tccatgatat cttgtaagtgtgtggcaaaa gaataaaata ttttgatttt 2100 atttaggaat aaatgtagat aaatatagtatttttgatat gtaaacttgt ataaaagttg 2160 ggaaagttgt ggttgataaa atctatccactacaaaaaaa aaaaaaaaaa acatttttga 2220 cgtcagtttt gaaatacttt aacgtcggtactaaagtgat cctaataata taacgtcagt 2280 tttgaaatta atcgactaaa tttgtgatagcaattaaaaa caatcctaag ccaaggttgt 2340 gtgtaatacg tgagtaaatc atatactcaaggtgactaaa ttagtggcac gatcgaactt 2400 aacatcctat gaacctatag tttgtttctttgataaacag atcacttata gcatagaaat 2460 atagaagaaa ataaagagaa gaaacaaaattgtaacaaga tccaagaatt ttgaatagac 2520 ttttactctt tatttaattt tgtgccttctcattaaaaga agggaagaat aaatttaatc 2580 gacattccca ctaaagaatc caaagtcaacttctcaaatc ttatggtaca ggaaaaatta 2640 tatgtaaagt gatgtataaa ttatgatttatcgcatggtg caccgcaatc atcaaaaaaa 2700 aatatggcca ccctttttat tatcttcttccacgtttccg ctatactctt acatttgtct 2760 tggtctttat tttgttttca atgccaaaaaaactttcact agcttcataa tctttttttt 2820 ttgttgaaca aagagcttca taatcttgaattcttgatca tgttttcttc aatagaacca 2880 atacatagaa aaataatctt tcttgctctcattttagcca caacattgat ttcaacgtta 2940 ttattttgct tacaacatca tcacatacataatactctaa ttttcattag atatacttaa 3000 ccattataaa tttcacataa caagtcgagatattaattaa tctaggcttc atttacggaa 3060 gaaaaaaaat cgataaagaa agcaacaacatgatactcat atactcctaa tgatgacaat 3120 catggagctt aggaaaataa cacatctctacttggtgttt caacttttta tgtttttgcc 3180 atttttaccc ctgtaccatt gccgccaaaatcagaaagtg ctctggggcc aataatacta 3240 tatggtcaaa tggactcaac gtacctttctccctttttct ttttgatagc gattgcgcca 3300 atataaaaag ggtatttcca gtggtggttctgtcaattag acaagatctc tttctcaatc 3360 tccaagataa gaaaaagtta agattatgatcccaatgtta tttgaaggtt aacacttaac 3420 actatagttt ggttttagag tttagtcaagttactaaaca cttcattgtt caatgtttct 3480 attcccaaac tccaacacac ggtatcttacatagaggtca tttgggacac cacgatttgg 3540 tccccttcaa gaaccttatt tcatttccaatattgattat tttcattgtt caatgtctca 3600 tatttttctg aaagaaaatg tatagctgattagtcataat cgtagcaaac ttcttgattt 3660 tgttatatgc ttagttagtc gttaatcgtctattcttcgg ctagcatatt tttcgggatt 3720 aatttagcca ttagtccaaa ttattcagacaacacgttct tgaaaactaa accttattaa 3780 tataatcacg aatgctttgg attcaatgtacttagtgaac catataacat ttgactgtag 3840 aaatttatag caatttactt tatcaatcctcccaaatcac tttatcaaag taatggggtc 3900 aaatgttaaa aattgaaaag ttatggagtgaaaccttaaa ttacataaat gctcttccaa 3960 ggggaagaag actaagagta tagaatctctctaaccagag gttctatcac caccaaaacc 4020 tgattgtaag aagccggttt cctccagatgactcacatca ccccaaactt ggatggtcca 4080 tttgtctccg tcgaataaac ggaacacattgaccgatgta ttgagtatct tttcaacttt 4140 tcttgcactt ggacgagctc tttcgtagagagatcggatc acacctccat gagtcactac 4200 tactatcctt tcaccttcat caaccaccaagtagacagaa accaatactt agaacatatg 4260 tgcagtgtag taaactgtga gaaagaaacacacagagaag aggaagaaac ctttatgttt 4320 gtcgccgatt ctctgtaatg cagttgtacatctatcgtaa agtttgtcaa gactttctcc 4380 tccacccttc attcacataa acgaagcaaaagttagttga atacagaaac caagcatcat 4440 tcatataact aagcttctta tcttactggaatatcaacgt ctgtgcggtt agatgaaaaa 4500 gcctagtaag cttccgggcg aattttcgaagcttcttgat acacaagccc ttgcatatct 4560 cctaaatgtc tttcccgcaa atcacgatcggtaagcacct agacaaccat tattcgtagt 4620 taaacttcta tgaagtctag aaagtcatatggattcagct tcaaaactta gatccctcta 4680 ctaaaaatca agcacaaagg tgaagactttagtgggtttg acacatagca ttatgtcttt 4740 agatgtctac ctcaagcttg ccgcatttagcagcaatgat ctgagcagtc tcaaaggctc 4800 tcttcaagtc agaagagtat acatgagatatcttctgctc cttcgataac cgctctgcaa 4860 ccttcatttc aaatgaaaga aatgttttaaaaggcatcat gatgattaag gttacaacac 4920 aatcaaaaga taacacatac tctttgtgcttgttgtcttc ctgcatcatt taactcaaca 4980 tccaaatgac cctgtaacac caaattataaccacatttca acatctctag tcaaaattcc 5040 caataactat acattcacaa agatgctaatcattactaaa tacaagataa agattcaaaa 5100 tttacgattt ctaagtcaag tttcgattccaaagatacag atacaaactc cattattgtt 5160 aaatttacct ggatttttct ctcggcattccaagatgttt caccatgacg aacaacaaca 5220 atctcagcat aatcaacatc ttcagaactgtagtaaaacc aacaaaggga tttcgtcaaa 5280 aacacgatca aagatgcaaa taacgagaagaagctagaag aagaagcata ctcgaatctt 5340 gattcctcca tgggagatgc tgaaatctgacaattgcttt agattcatcc aacgacgaga 5400 aaccaattcc attgatatga cattaacactaactgggctt tacttagatt ttgtttatgg 5460 gccttgccct ttgttcgaac gaagcaaatatgttttcgtt ttttttcctc tctcacgtta 5520 aaatcaataa caaactttat tacatactactacagtacaa cttctataaa ttaatactcg 5580 ttaaattaat aatctctata aattaattaattattccagt cccgaattga aatcaatata 5640 aaaattgata taactcgata aattaataagattctaattt ttaaaaattc ttatataaat 5700 atatggtcca atcaatagta taaattaataataatataaa tttaccaata tatattataa 5760 atatcatatg taaaatatga cattgtttttcttcaatttt tattattttt tttgtatgtt 5820 caattctaat attttacaac ataaaaaaaaattgtattgt ttttaaactt aataattaat 5880 ttgtattttt aattcgtttc ataaatattattgattataa aaaaaaaaac ttcattttat 5940 atcaaatttt tgtaagaatt tacaaaatttgtacaatttt aattaattta tcgataactt 6000 aatatctctc taaattaata aatttttttggtctcaatat tattaattta tagaggtttt 6060 actgtagttg gttaatttca tgaaaacatgtatggagaaa taaaatagta acataacaaa 6120 aacttgctac tgcatccgcc atatctggatctctaactgt agctgaagag aggccaaagc 6180 aaatacgtaa aactctgcta aatttcttataaaatatttg gaacgaagca aataatattt 6240 aattattaca atgatattta gctaactatatattaacctg cggtataccg cggtaggctt 6300 atattttagt taaactaaaa ctttttattgtaaagatgat ttataaatag ataattttat 6360 tttatttgat tttaaatgta tagtaaactgcgagttgtat atgttttgtt gatattattt 6420 atattgttta gtgtttaaga ttatacacttgtattttaat tgttaatttt agagtttcac 6480 ctgtagtata ccatcttcta ttaatatcgatcttaacccg tcaattctag gattttccag 6540 cttgtattaa aaattgaatc acatcatacacataaaaaaa tctaatatgt tattaattat 6600 tgttgtatat aagattataa attttcaaaataatatgtat gaaattgaat ataaatattc 6660 aaatgatgac ccattactca gtagaaagttttcttaaatc tatttttgac tgttgtaata 6720 ttttttttat gtattgaaca gtttatatttgtttttaaaa attcaaataa tggcatatgc 6780 gaaaaaaaac tctaattatt tttttataacgatgatatta ttttttgcaa aattagaatc 6840 atataaagat gagaggtgaa ctataataattaataaaaaa ttaatatgat aatttagata 6900 tcaaatctaa tttgtttatt ttaattggttacttttttgg aaaataataa tgtattttgt 6960 ttttctaatt aaattaaatt aattaaaatttagatatcaa atcttatatg ttgattttga 7020 ttggttactt ttttggaagc taatatatatatttgttgtt tttggataat catgaattca 7080 gttttatcta gattttgttt tcaaaaatattaactaacct gaatattttt tggtactata 7140 taagtaaaat tgcgtggtag gaaattttgacctaaatctg tgttgatcaa ttttaggttc 7200 tttctcaaaa cgcattttac aagtagggaatagaatttga agaagcattg tgtttaggtt 7260 aaaaactgaa tctctctatt catcttccttttttattttc atataaatat atatggttta 7320 gaaatcaggt gatgttcact tgattagattcatcttcttt ttatacattg aggtttgtgt 7380 tttcatcttc tcactttctc taacatatctgagtgtacaa cgcaatccac gattttgatg 7440 gctattctca ttactatatt atttagaaaccttaatctct ttactgttta gattcacata 7500 aaaattcgta cgttgtgatt tttttttttttcagttcgtg tatagaccta ggcatacgat 7560 tttccctttt gttctttttt tttcatgtttcggattttcg tgtttaagaa aaagaatctg 7620 taaatatgtt tgtttgattc agtttaaaaggaaaaaatta catcatgtat ttcgatatat 7680 cctatattta aatgtatttt gtatatattctagaaattat caaaaaggta taatttcatc 7740 ttctttcttt ctttcacaga ttatgatgtttcatcaaatg gacaaaatat cgacaccatg 7800 cgcggcatgc aagcacctga ggagaaaatgcacagaagat tgtgtgtttg cgccttactt 7860 cccctcgact aaactggaca actacgaagcggttcacaag gtatttggag caagccatgt 7920 agctacactt atcaacagtc ttcacccatgccaaagagaa tttgccatgg acacgctcgc 7980 ttgggaggca caagttcaag ccaatgatccggtcaatggc tgtctgggta tcatttacaa 8040 cctccttagt cagattaaag acttggaagaacaactcgcg atcgtcaaga acgaacttgc 8100 ctcttactgt atcgtcccca catttgtgccaccaccttcg atgacgaatc tggaaatgca 8160 taacaatcct atgatgatac cggaacacacacctaataac ggtggttgtt taacgggtca 8220 acagttgtat aacgaggctc aacgttttgcatccaccaca caaagcgctc aaatgcaaga 8280 gacgcagacg cagcacaatg agagttaccgtgacaaaagt tcttatcaaa aatttggacc 8340 atgctttaac ctacattaat cattatcatattgaatcttg ataacaatgg taagctttta 8400 aaatctttta agtcttgtct agtcgtttgtataactacat taagtgccca atgcctttta 8460 atttattcta aactctttta ttatcacttttgtttcgttt acaattaaaa cttctctttt 8520 atgtgttttg aatttctcag cataatggtatgaactgtca taattcttta tactgtatga 8580 tggttctctt ttctttggtg tgggaaatatatgtcttttt acgtgattga atatataaat 8640 actattattg tttttagtta acaaaacatccgttatcgtc cagcggttag gatatctggc 8700 tttcacccag gagacccggg ttcgattcccggtaacggag ctttttttgg tttttaagat 8760 tcgaaaaaga actttttgcc tcatcactatctttacgtta ataaactcag cggtagtcca 8820 cgccgtaaac cttaacattt ctaatccggatctttagtca atttgtttat ttcttttaca 8880 aacactccca acattcaata aggatcttgtgtagaaacta cactctacac aagtgcacaa 8940 cgccataagg cccataacaa aaaataaaggtaaaataaag gtgcacttgt agttatgtac 9000 aaacgactag acaagagtca taaaattagaaaagtttaat gtaacattgt tatcaaaact 9060 attgtttgta tggctgcaag aaaaataaacatcttatgat ctataagaag cttttttttg 9120 ttaatgacaa aaacaatttt tgacttggctcaaaactacg atcacaaatt tacaatcgta 9180 acaacccgtc ttgtgagact cagttggtgttgcaacacat caaccctaac ctcttttttt 9240 gtgaattctt ctgtcatacc atgtaggttattggttcgct tgacgttcct caaacccaag 9300 acctcaccct ttaacaatct ttccacagaacaagctgtta ccaattaatc tgccaaaaag 9360 acccacataa aagaagttag ggttatgcgtagcgtctgct ggatccgacg aggcggattg 9420 ttacaacaat cacccacatc aaattgggatattttagaag taaacaagta caagttaagg 9480 taaaacaata catatacatg agaattcaattaatttaaaa aagacataac aatttaccca 9540 cctcacaatt cgtactcatt caattacaaattttaggagc aaccaaattg tgttgataat 9600 atagaacagt aaaattaata ttgataccaaaaaaaaaaag atgcagttgt atcatcaaaa 9660 cgcggcggcc tttgtcgtat caaatcagataactaaaaca aataaaacgg tgtcgttgca 9720 aacatctctc tcctcttatc taacttagagagcgaccaat aattgccttt gtctttatca 9780 ttcgtcgttg atccatcttt ttcccccaaattcatatcct tccttagata tttttctcct 9840 tcttcttctt cttctagatt ccagctactccagaagattc ttcgacttaa tctgatgtga 9900 ttaggaagag taatagagca agagaagaatcagaaaaaat ggatccggcg actaattcac 9960 cgattatgcc gattgattta ccgattatgcacgacagtga tcgttacgac ttcgttaaag 10020 atattggctc tggtaatttc ggcgttgctcgtctcatgac cgatagagtc accaaggagc 10080 ttgttgctgt taaatacatc gagagaggagaaaaggtttc ggtttttttt ttggaatgaa 10140 aaaaaaagat ttgatattta taggctttagtgattgattg atgatcgggt tttttagttt 10200 ccagatttag tagctttcct ttttagagcttgttgatcca gtcgtcttta atggtggtta 10260 cttactgtta ctatgattat taatgctttttgaaaagttt tgatatttct cagtaaatga 10320 tgataccttt tttttcgtgt gttttgcagattgatgaaaa tgttcagagg gagattatca 10380 atcatagatc attgagacat cctaatattgtcaggtttaa agaggtatat ataaatacta 10440 caccttgatg tttcttctgt gtgtttttgtgttataagtc agtgtggttt agagtttgtt 10500 tttgtattgg tttaggtgat tttgacgccttcccatttgg ctattgttat ggaatatgct 10560 gctggtggag aactttatga gcggatttgtaatgccggac ggtttagtga agatgaggtc 10620 tggattctga ttcatcttac tgtgtttcagtattgatgtg tgattactga tcttttgtgt 10680 ttttttatgt ataggctcgg ttcttctttcagcagcttat atctggagtt agctattgtc 10740 atgcaatggt atgcagagct gtgttcttgcaacttacttg aacttttctc gttctcgtgt 10800 ttattactct ggatttaatt ctgtgtttgattttcctgtg aagcaaatat gccatcggga 10860 tctgaagctg gaaaatacat tgttggacggaagtccggca cctcgtttaa aaatatgtga 10920 ttttggatat tccaaggtac tatttctctcaaggtttgtt ttctgaattc tgagtttata 10980 tatgctactg tatggccaag cgcagaacagcgggaacatc ttgtgattcc tcattgtgct 11040 tcttatcgtt catgctcttt acctacatgtgtttcctact aagtgtctgt ttttgctttg 11100 atttctgctc aagtattaac tatgcgtttcttttcttcag tcttctgttc ttcattccca 11160 accaaagtca actgttggta ctcctgcatacattgcacca gagattcttc ttcgacagga 11220 atatgatggc aaggtaaggc catgagaccaattcctctgt ctagagttaa aaccagctta 11280 tccatatgac tgtttatacc ataaatattctttgagttgt cctctgaaat agctggtgtt 11340 acatttttcc agcttgcaga tgtatggtcttgcggtgtaa cattatatgt aatgttggtt 11400 ggagcttatc cattcgagga tccacaagagccacgagatt atcgaaagac aatacaagta 11460 atgtttttta attgttcttg atgtctcattcatcatcatc cacaaccttg ttatactcat 11520 tatccttcaa aagttgattg tttatgtttgcttctccatt gattttaaaa cgcagagaat 11580 ccttagtgtc acatactcga tcccagaggacttacacctt tcaccagaat gtcgccatct 11640 gatatcgagg attttcgtgg ccgatcctgcaacagtaagt tttacacttg aaacaaactt 11700 tgttcgttaa ggcattttga gttctaaatagacaaactga gagacctcaa tgtggcagag 11760 aataactatt ccggagatca catccgataaatggttcttg aagaatcttc caggtgattt 11820 gatggatgag aacagaatgg gaagtcagtttcaagagcct gagcagccaa tgcagagcct 11880 tgacacgatt atgcagataa tatcggaggctacgattccg actgttcgta atcgttgcct 11940 cgatgatttc atggcggata atcttgatctagacgatgac atggatgact ttgattccga 12000 atctgagatt gatgttgaca gtagtggagagatagtttat gctctctgag attcctgagg 12060 acaaagtctg ttttgtccgt actgttgagacacaccagtg gagttttgtc ttagctccac 12120 acactccacc gttcattttt ggatcgtttgttgtttttta ctctacaagc tttggattca 12180 catacatatg tattgtaatg taatatgtaatatattttat gtatttttct ttgttttaat 12240 aactattggc acattttata caaatgatatggtactagat ctgaaaaaaa aaaaggaatt 12300 ctccaaatta taacaaagtt gaaatctacattttacatta ttcatatgag tgatttgaca 12360 caattggtac atcaagaatc atcacttcacgacgtatcgg atcgcatcgc acgcctttgt 12420 tatgtttttt ggaactccag ccgatgtggatatcaatctt cggtttcggt ctgtttcttc 12480 gacaatatct tgattagtac tctgcgatgtgctacaattg ctgtttttgt taccgtaact 12540 tcctacgaaa gagtgtgaat aagagatgtatgggccgctt gataacgaag tagtatggaa 12600 tcagaagcat gtacgcggta acgaaccgttgaaaagtgtg aaagggattt ttaaagaaaa 12660 ttagcaaacg gtatgtgttg gaaacatacatatattaata aataataaaa tattatatca 12720 tatttaaaat ataattaatc tcacaaacaatacatctaca tatatagtca ttttaacaaa 12780 taaatcctgg atttggaact tatttactactcaatgccat taacattaaa tcttttctcc 12840 aaaatgatta attttactat aaatttcttgatttaaattg tcaaccacat tatcaatcaa 12900 atttaatcta aacaaacttc aaatcaatcccttaaaatta gcataaacat atttcttaat 12960 aatattttaa aacaaagttt tttgttaaaacaaatattgt tcttactaaa cgttttttgt 13020 ttaaataaat aaatcatgta tttgaacttatttacaatat catgatactg acattaatta 13080 tttaggaatg aaaatattta attaaagatttaaatcttct aaatccttac ttttagagtt 13140 attatgtcat tacctaaaag gctcaaaatataaaaatttt aaatattatt atttatatat 13200 ttaaacttta taaaacgtta ttaacatttaaacttataaa atttttaata tatcataatt 13260 ttttaacatc attatataat atatagagtttaagaaaatc ttaatctttc cttcatattt 13320 tgtgactcga aattttaaaa atgaacatatattaaccaat tggcgaaaaa tgtgtgcgtt 13380 caacgtccat atcaactaaa atatttvgagcatgattcat aaacatatca taaataagat 13440 taacattaat aaaataatgg tttttttacgggacgggttt ggcgggacgg gtttgacvgg 13500 acgttactta ataacaattg aaaactataaaataaaaata ttttataaat cgatacaatt 13560 tacaaaattt tacatatact aactttaaaatataaattgt cttcgcgatg taccgcgggt 13620 taaaatctag tgatcattaa taattcaactaaaagtaaca aaattaaata gtcttttact 13680 ttttttatag ataattaaat caaatagtttaaagtcaatt ttagatcatt gtcagtaaaa 13740 aaacatcatt aaactcaagt ctttcaaagttaatttaatt caatttatgc aaaaaaaaat 13800 catgaaacat agatctcaaa agaagcgaaataaaaagatt attgttaatt attattttga 13860 taaaattaca catagattga gaaagagtttttcaataatt atggggaata agagagagag 13920 agagagaaat agatttccga aattgattacaagaagaaat aatttcaaca aagtctctgt 13980 ttttttttat caagctctta ttttactacaagcagaaata acttcagcaa gtttagtgtc 14040 catttcagat atcttggcct tgatttctttgatatcgtcc cattgatcaa cttgaaaatc 14100 cacaactatt atgcttctta atgcaagttctatcgtttcg attgctttga tgtacctaaa 14160 gataaacaga acaaacataa tactcgtgttatttttccac aacatgatag gttttgtacg 14220 tttagtgttt ggagattatc gaaatcatgtaaaaaaaatt gttacaaaga agaagatatt 14280 tttctctaaa ccattaaact aagaaattaggcgatccaaa aaccaataga aattcatgtc 14340 atatatacga acctgtactt cctgagtacttgtctgcgtc tgagtttcgg ataagcctca 14400 acaaaaacat aagctaattc aaggaaacacgtgagacgtt cgttgacttc ccttaatggt 14460 gaaccttcca ctttactccg cttatcgatttgaaccaaaa acggctcgat actaaggatt 14520 gtagcgcaga gacggtgtac gaaagatatatctcttgctt ttctgaaccg gtcgtaaatg 14580 gcttgggctc caactccaag aacagcccctatcgcaagct caccaatcgg cattttttga 14640 aagtagttgt ttagctctcg aggtgaatatagaggaatct atgtacatgg aaggatggaa 14700 ccatattaaa tagttttatg tttaacaagttaacgagtgg ttttaattat atgaagacaa 14760 ttcaagagat tgactcatag acttagtactgtacgggtca acaactctct ctttttctag 14820 gtaagaggag atcgttggat ctatatgcaagttgtcgtga gtattaaatt acgtagaata 14880 ttattgaatt acgtcgaaga agcgagagtcaatctcactc tcaatggtta acttgtacat 14940 ttagaagaag gaaaaatcaa cgaagttggctgagtaagaa gtgaagaaga aaaacagtga 15000 agaaagccaa aaagcagaag aggaaaatggtggtatcaac taaaaatatt tcaacaaagg 15060 aagttactac taaaaatatt tcaacaaaagaagttactac taaaaataaa tactttgcat 15120 gttgcagtat atatttaaaa tttagaaataattatatcta ttaaaaaatc attttgtaac 15180 agatgttcga ttatgatata tagaattattttgtagacgt tttataaaat agtttaaaaa 15240 attatattga agatatgaga tgaaccacaatacgtatttt tatttttcgt attttcaaat 15300 aaactcttat tattatatga aatctgaattagcccagaat attattagat ttggtttata 15360 atttaatctc aaaattttct tccaaactgaaaacagaaaa aaaaaaaaaa aaaaaaagaa 15420 gaagaagaag aagaagttaa aaaccactaatctgaaagat ccactctaat ttgtataaat 15480 ttttcgtttt aagttcaaag atgggatcaaatcaaatgag aagaatcctt aaaaactttc 15540 atctttatgt aagaagcaaa agcaaatttagttaagcttt tttctaagtt ctttatatct 15600 tctttcagca ttaattcatt atccacaactttgttatact cattatcctt caaacttgat 15660 tgtattgagt ttgcttctcc gttgatcctaatacgctaag ttcaactctt tgtaacaact 15720 ttgttcttta aagcattttg agttctaaataaacaaattg agagaccaat gtggcagata 15780 atcgtcattt tgagatcgtt tgttgttttttactctacaa actttggatt cacatacata 15840 tatatatata tatatataga tatatatatatatatatatt gtaatgtaat gtatagtata 15900 tttctgaatt tctctttgtt taataaccattggcacattt atttattttc aaagtatgtc 15960 attagattat tcatattaat acatatatatgagtcgtttg acacaattgg gacatcaaga 16020 atcatcactg cagaacgtaa atcggatcgcacggtttgtt tttcctgaac accagccgat 16080 gtggatttca aacttcggtt gaggcattattttgtcaagt ttagtgctga tttcagacat 16140 cttggccttg agttctttga tatcagccaattgattaact tgaacatcca catctaccac 16200 ccatcgtaat ttagcttcga actctttgatatctctcatg tacctaaaga taaacaacac 16260 aaatataata cacatgttat tgacttaattcatagtaaat gttaggtttt gatagattta 16320 gtactgttgg gagtttatgg aaatcacatataggaactat ttagcacaaa cctgaacttc 16380 ttgcgtacgt ttctgcgtct cagctccgcattctcctcaa caagagaaac agcgttctca 16440 aggagaagct taagacgttt attgactttcctcgatgttg aatcttccat ttcttcactg 16500 aacttatcaa tttgaaccac caacggtgtgatactatcga ttgtagcttc gagacggtgt 16560 aagatgaatc ttgtggttac agatctatcttttgctcttt tgacggcctc gtggaggaca 16620 ctgagagcaa gtccaagagc accccctgcggcaacctcag caatcatttt cttgaaatta 16680 gtttgttagc tctcgaggtg aagagtttttgatgagttat attgatgata ttattttgtt 16740 tggtaagaaa aatataagac catctattatattatataga ggtgaatatt tataattcct 16800 ttttcttctc aaatatttgg taaagtgttgctctattaat tcacataatg ttagtattat 16860 acacaaatat tataagggtg aatgcaatgagaaatctatg aacatggaag tcttttgctt 16920 aacaattaag ccgtgtagtt tgtataaagtcaaacggatg ttctttgttt ccgtaacttc 16980 ctacgaaaga gtgtgaataa gagatgtgtggaccgcttgg taaagtacca tgcagttaga 17040 agcatgtacg gggtagtgaa acgtcgatttttattataaa ataaaataat aaacgatatg 17100 tgttggaggc gtatatatat taataaatagttaaataaca aaattaaatc gtcttttact 17160 ttttttatag ctaataaaat caaatagtttaaagtcaatt ttagatcatt gtcagtaaaa 17220 acatcattaa actcaagtct ttcaaagttaatttaattaa atttatgcag aaaattcata 17280 aaacatagat ctcaaaagaa gcaaaataaaaagattattg ttaattatta ttttgataaa 17340 attacacata gattgagaaa gagtttttcaatcattattg ggaagaaggg aggaagaaaa 17400 gaaaaaacag atttctgaaa ttgattataagaagaaataa tttcaacagt ctctgttttt 17460 ttaaatcaag ttcttatttt attacaaagtgaaataattt cagatatctt ggccttgatt 17520 tctttggtat ctttctaaaa aacaaatttagagaccaatg tggcagagaa tcgtcatttt 17580 gggatcgttt gttgtttttt actctacaaactttggattc acatacatat tatatgtatt 17640 gtaatgtaat gagtaatata tttctgaatgtctctttgtt tacgttacat tggcacattt 17700 atgaagacaa aagacgtttt tgattaattatattgatgat atatataaag acaaaagacg 17760 tttcacaaaa tattaaaacc ttaggaaagacaccccattt atcatcaatg gaggtgctct 17820 tagataacaa tctagaatcc ttatcgctttagacagctgt gttattgact agtcatcatc 17880 taaagaggat aaggattgga aacgatttgaattggagacc aagtgcttgg agagtaagct 17940 tagggttgtc tttgtatgtg tgtatatatactcctcaaga tcgatcaata acatcaagca 18000 ctttttcaac cattcttagt ctttacaattaatgtacgaa gaggattatt atttattaaa 18060 ttacgaaaaa gaagtgaaaa tcgatctaaatgattgactt tttacgtaga atcgtcgaat 18120 tgcatgtaca tttccaccga aattccaaaaatctgaatta caaataagtt ggaaccgatc 18180 gatcttgttt tgtatattta cgtacaaggcagacgtacat acatgtagtt tggattatca 18240 tatgtatgat caacgcaatt ttcgtgaatagaaacgtgaa tactaacaat ttcggtgaat 18300 acctaccgta aatactaaca ttaaaatctatgacttctta aaataataat caatcaaact 18360 tttacatttg attttatatt ttcctcagtttttaggccta tgatacacct gccttctcaa 18420 aatattagtt ccgtgatgtt tgctccatctaaggtggata tcgatcttcg gagcatccag 18480 ctctcgcttc caatgatgga tcaacgctttcaactatgtt ctgattagta ttatcctcat 18540 cagatctaat acagtatatt ggttagtattcatttcaaac atcttgccca tgagttaacg 18600 ttcattaata tctgcccact tttgtatgttattacatgaa aacaaaaata cgataggttc 18660 tgaattacgc tgctgctaaa atacacatgcagcttgtaaa tgtgcggcgt aaccttatat 18720 gtaatgttgg ttggagcata tccatttgaagatccacaag gccaatagat tatcgaaaga 18780 caatacaagt aaagcttctt ttctaagttctttctatctc attcattatc ctaaatcgca 18840 gagaatcctt agtaccatat actaaatcccagagtactta catctttcac cagaatgtcg 18900 acatctaata tcgagggttt tcgtggccgatcctgctaca ataagttcat tttatataca 18960 aatgatagca ttacacctga ggaaaataaggaattctcct aattatttca aagttgagtc 19020 tacattttaa attaagtcct actatcacatttgagtgatt tgacacaatt ggtacatcaa 19080 gaatcatcac ttcaagacaa atcggatctcacggtctttg ttccgttttc ttgaactcca 19140 atgaatgtgg atatcgatct tcggtttagacccatcactc gaacattcaa caatttcttc 19200 taaagatcgg tctgtttctt cgacaatattttgactacta ctttgtgatg tgctatgatt 19260 gctcttgaaa caaatacaat cagttggttgacgagtgatt tcatcaagtt tagtgttcat 19320 ttcggacatc ttggccataa gttctttgatatctacccat tgattgactt ggacatccac 19380 atctaccatc catcttaatg aagcttctaactctttgatc cttctcttat acctaaagat 19440 caaaataaca caaaaatatt aatactcatgttatttcatt aaaatgataa gttttgttag 19500 atttatattg agaaattacg aaaatcacttatagaaatgc tcatctaaaa aaagatttca 19560 cttgaaaaaa tcttgaatcg aacttgaagtaaaccgttcc tagctaatcc aagatatctt 19620 attttaaaac accatgaaat taatcactgtaagtcagtat ctcaatctga gattctaatg 19680 gtatccatct tttaagtagt gacaaaacaaacaatttttt attttatttt taactattaa 19740 atttaagaaa taagaagact tcgtatgaaccaaaagaatt catgcatttc atggattgta 19800 tacaaacctg aacttcttga gtaagtttctgcgtctgagt tccgcataag cctcaacaag 19860 agaaacagcc ttttcaagga gatgcttaagatcttcgatg actttcctcg gtgaatcttc 19920 cacttcttcg ctaagcttat cgacttgagtcaccaacgga gtgatcctaa agattgtagc 19980 atcaagacgg tccaagatgc atcttgtagttagagatcta tcttttgctt ttttgatggc 20040 atcgtgaagg acttggagag caagtccaagagctgcccct gccataatct cactaaccgg 20100 catttttctt tgggtcaaga gattagctctttagctctca acgtgattgt atggagcaat 20160 ctatatacat gaaaccattg aagaatcttttgtttaacaa aacgttttgt ttttgatgaa 20220 ctatatgaag acaagagacg ttgacttaaagacttagaat tagtcgactc tctttctttc 20280 taggtgaaag gagataattg gatctatatgcaagttgttg tgatttttaa attacgaaga 20340 agggagaatc aatctcaatg attgactttaacagtaggat taaatgttga cttgtacatt 20400 tgcattaaaa aaatccaaaa atcttaatttacatattagt tggaaccaat cttctttttt 20460 gtttatacaa gcaggcgtta tatgtatgataagttgataa cacaatattc gtaagtataa 20520 tttttttttt tttttacatt tgattattttctttctcaat tttgctggtc tatgacacat 20580 accacatcaa aaaaaaaaac taattcaaaacgaagcgaat tccgtgatct ttgctttgct 20640 tgctccatcg aaggtggata tcgatcttcggcttagaatc gctatttgag catccagctt 20700 ttgcttccaa tgatggatca actctttcgactatgtccat attagtatta tcttcagata 20760 taatacaatc tattggttga cccatgattttttcaagttt agtgttcatt tcagacatct 20820 tggccataag atctttgata tctgcccattgattgacttt aacgtcaaca tctaccatcc 20880 atttcaatga gccttctaac tctttgattcttctcttgta cctaaaacaa cagtacactt 20940 tagtatgtaa ttaaatacat acatgaaacaaaaaaatgat aggtttttgt ttaattggtg 21000 tctatataat gttgggagat caagtaatatgtagaagtat tcatgtatct aagattaaaa 21060 aaaaaaatca ttatttcatg tatatatatacgagaccaaa agattcacaa acctatattt 21120 ttcaagtaag tttctgcgtt tgagctccgcataagcttcg acaagaacca cggctttctc 21180 gagaagatgt ttaagatctt cgaagactttcctcagagat tcatcggatt ctttgttgag 21240 cttttcgacc ttagccatca gtggagtgatcctcaagatt gtagaatcga gacggtccaa 21300 gatacaactt gtggttaagg atctatcttttgctctttgg atggcctcgt gaaggatttg 21360 gagagcaagt ccaagagcag cccctgcgataatctcggta agaggcattt tgtatagttt 21420 tttttaaagg attacttaaa ttacaaagagatttattgca attagcttta gctctcgatc 21480 gcacgaggtt agtatatgaa tgaatcaatattgaattggt cttttgacaa aaacacttat 21540 ttggattgat tttttccaac ttgttgtgattatggaatta aagaaaaagg agtagagaaa 21600 tagtctcaat gtggattaat tttttctgttaaatattaat attttgatca acttgttgtg 21660 actatgaaat taaagaagag aagagcgagcagtgaagcat tctcaatgaa aattaatttg 21720 tttgtttcaa gttatatatg aaagtaaagaaaaaaaggga agataatcat attcgaaatg 21780 atcgaatttg tacatacgtt ggattaatcactgacgtgta ctgaaatctc ctaacgtttc 21840 tgtccattct cgtttttcta atttatattggtagaaatat tgtatattct atattgacat 21900 tatcggtgaa atattatgat gactatcatatggaaactct tttagctaag tttagattaa 21960 atatagaaaa agtttttctt tcatattgatcctactaatt atagtgaaat gtttcgctta 22020 cctctttgtt aagtttggtt tacagctagtagttgttaaa gcaagtgaaa attttatgtt 22080 tccgcatttg attgtgcgtt tgtgacccataagaaggatt tacaagaagt ttggttagat 22140 aaaaatcttt tataataaga gaaagttttttctaactttg cctaccaatg cgatatttga 22200 cactttactt tgatgacacg tgtacttatcattaattata atttaactaa aataatttat 22260 taatagatat atcttatatt tagaaaaatagtcattaaac tatgaaatta ctacatatta 22320 aactacaaat tttgaaatga atttttttctaccataactg caactccctt aactaattac 22380 aaaatcaaat ctttaaaatt ttgacatgatattatgtttt agagtacaaa gtaatcacta 22440 tttatatgtt tttgataata aaaaattattatatttacta ttatagatta ccattaatgc 22500 atactcttaa cattttctta cctgcttaaccgtaataagg tattaaacac tatttaggag 22560 tactttgatc caaaaattta tgaccctttatatctcataa gttacaatat tttgatcacc 22620 acattaaagt acaatttatg acttaatatataactaaaaa tggcttaaga atgcaactat 22680 ataacaagca tggcttgaga aaaagcaattggcaacctca agtgtctcaa atgctcagct 22740 ccaaatacaa tatccgtttt tcatttttttttgcatattt tagttgtaca tcgatatatc 22800 tttctagagt ttggatgtgt ttcttacaagttcagtaaac atctaagctc tacactaact 22860 tatatttgga tatagtatat tctttgaaaattcaaattct caatttcaca acaattatgg 22920 gatatgctag ctaaataaat atttggtaaatacatgcaaa gttttctctg gtgttgttaa 22980 agtccattga gatgcataat tattcttgctagaataatca aatccgaaag tacctactca 23040 ttatccgtga ttgtgtatct ccatttgttttttctgtcta ggcaaggaag acaaagaaga 23100 aggttgaaga agttcatgaa atagagggaaattcttagac tatgaatact aaagattacc 23160 atatagaaaa gagtatttac ttgtttttttggtctacttc ctttatttat ttaagtttga 23220 caatgttaat tgaaacctta ttttcatgaaatagagggaa attcttagac tatgattact 23280 aaagattact atatagaaaa aagtatttatttgtcatctt agtttacttc cttttttttt 23340 ttttaagttt gacaatgtta attgaaaccttatttagata ttcactaaac aaaattaagt 23400 tgtttcctca cagtaccaag acaaccaaggggttccgaac caactataaa acgattaatt 23460 tatttaaaat gtagttaaac attttaatgtttatttagtc ataaatatta tatatatata 23520 tatatatata tatatatata tatatatatattattaaatt attttataac attttttaac 23580 tttgcctacc aatgcgacat ttggtactctactttaatga cacgtgtact tatcattaat 23640 tgtaatttga ctaaaataat tttttaatagatatatcata tatttagaaa aatagtcatt 23700 aaactatgaa attactacat attaaactacaattttgaaa tgaatttttt ctaccataat 23760 tgcaactccc ttaactaatt acaaaatcaaatctttaaaa ttttgacatg atattatgtt 23820 ttatactaca aagtaatagt tcactactgtaaataattct ccaattttac aagaattttt 23880 ccatctttag ttttttttat accataattttataaatttt accatgttct aattttaaat 23940 cagaaaatcc tctaaaagtt taactattagttacatttct ttgttctcct tattttaact 24000 tctcgaatca tattaacata gtttttgtaacatcagtttc ttctctttag ctttttttac 24060 acaagaagtt ttttgtcttg tgtcaaaagctgatcatgaa ttggagagaa gtaataatga 24120 ttccttgatg agttagcgta atgccaacagtcgttgccaa tagacagcat tatccacaga 24180 aaacatgtat tttacataat ttagggtagaaaatatatat acaaactttt cactcatttg 24240 ctataggtag ttaatttatt ttgtcctaccacatacaaat ctacattacg ctaccaaaat 24300 atgctaaatc tttttatcca tataagaagtttaattttta ataattagat atcttaatca 24360 aatttctagc gtcatgttca aaaccgatgatcataagcta tgtcttccat cacaatgtcg 24420 aatgacacta tacatggaat catgtgtcgtgtgaaatcgt agatcataac attcttaacc 24480 aagactcaag ttaatctcta tctcagaaccttgtgggttg ggcgtaaaac atctcggggt 24540 tggctcatct cattgggcaa agagaaccccaatttatttt taggactttt cttttcccgt 24600 tcccccacct cagtatagtg cttcgcttccggttcttcgg aagaatcaac ttacctctat 24660 ctttttatta tataaaattg tgttttaccgatataacaac ataagatata tatatacatg 24720 aatatagtgg aaaatgagag agtgattatttataagatgg aaatgaaaaa ttgataaacg 24780 taaataaacg taaatattac aaataatttgaataaaaggg ttttagtcaa gtaaaaaggt 24840 tttccaagag acatcttaaa tcatatttttaattactttc gagttatata ttaataatta 24900 tagataaatt atatatataa tttgtccttttttgtacatt tctcatattt tatatgactt 24960 tttttttcct ctgaactgac ttttttcctacgaagaaggt tcatctattt ttttttccaa 25020 gacaattggt aatacatgca tttgcattttcacaaaaaca aatacttaat atttaagttt 25080 ttttaatagt aaaacatact gttttcacaaaacaaaaaaa gcaattgtgt gcataatttt 25140 taatcaacta atgtgtcttt ctaaacacatcatactttgt aaacatcaaa attactatta 25200 ttaattatta attattttga gcgatatattaacaattata aataaatttt atatctaatt 25260 taatataaat attattctat attcaaaattaaaaccgaat atataattca cccatcgcgt 25320 ggacaacttc tagtaatata atataatagagattcagata gataatagtt ggttcttaac 25380 caaatttcaa ctataataga gtacttatgatgcatcatgc acatttgtat ctcatacgaa 25440 taagaaacta agtgtgcaaa ttgatatgtatccatgcaga gttagtttgg attttggatg 25500 atttttatct catagtcata gtcaattgtctcgcaatcaa tttatgaaga catatcaaat 25560 gttgtcaaat catttgtgta tgaacatacgttacgtgatg ttcaatctaa taagagatat 25620 tgtatataaa aggcacaaga gttatttgtcttgtcaacca ttggaatgtt ctcagaagtt 25680 tataataaca taaggttgtg aactaattaatatattagtt ataccatttc cagtaataac 25740 aatataaaat gcttaacaaa agtaataagataaaatgttg gtaaattaat acttacatca 25800 cacattcacc tacaataatt ccaacactccaaaaactata tcacatttgt tgagtttttt 25860 ttttctcaaa aaacattttg tggtctaggacacgatcgtt acattaacaa atcatttgaa 25920 gatgaaccgg atctcgtgct ctttggtctggtttttccat cgaaagtgta tatcaatcgt 25980 tggctttgtg catccatctt cttcagaagatcgaccagtt gcttcgatga tatccattgg 26040 ttgacgcgta atatcatcga gtttagtgttcatttcagac atcttaccca taagtttttt 26100 gatatccagc cattgattga cttgaacatccacatctatc atccatctca aagaagagtc 26160 caactccttg attcttctct tgtacctaaagaaagattca caacaacaac aaaaaaattg 26220 catgaaaaat aaaagatttt ggtaggatatttttttgtta aagtatatac tttggttggt 26280 ataatttaac actacattac atttacatatggaaatgatt aacgacattc acaaacctat 26340 atttcctgag taagtttcgg agtttaagctccgcataagc atcaacgaga cgaatggctt 26400 tctcgagaag acgtttaagc tcttcgatgacttttctgaa aggttcatcg acttcttcgg 26460 tgagggtatc gatcttaatg acaaatggagttatcttatg gagtgtagca tcgagacggt 26520 ccaatataca tcttgtgatt aaggttttctccttggctct tatgatggcc tcgtgaagga 26580 gttggaggga caatccaaga gcagcacttgtaagaagctc aacaagaggc attttcttca 26640 gaaaagcttt aaagctttga ttaaatggagactaatcgat attatagaag gaatttaagg 26700 acgtggttgg ctttaacttc ttgccggaggttaattatcg tatgactcta tgtggaaatg 26760 gtctttgtta acaaataatt ttatttctaagtaaatacac atacttgaat taactttttc 26820 aaattgctct aattttgtaa ttttagaatcttttgacttt gtgtagttaa gataactttc 26880 ttcaagttgt tgtgaactaa tgttagtatatattctcatg tatcccgttg aacaaaaaaa 26940 aaatatcatg tgtccaacac acagacgcacctatagcgcg tgtaatacac caccagaaat 27000 ctccaaaatt cagggaaatc gagttcttttttaagcgcgt ggtcggaaat tcagggaaac 27060 attccttttg ggcttttaaa aactattaaatgggccttag gttgtgctgt atgcaactta 27120 atcaaggtag gaaggatcaa atcttggatacatggtgatt gtttttgtct attaagtgaa 27180 acgcaaagca acgatcatgg atcaggaacactattttgaa cggtatggtc atcaaatctt 27240 acttgtgaca caggctgcaa aatcccaaattgggaaagtc ccaccattct aaagtagatg 27300 tagacccttg aggtaaacat gtgctattcattgtggaaag tgatatttat cttgttctca 27360 tcttctctat cattgattgt agaagcatctgatgaggaaa gaggttgtgg taggaccttg 27420 cttctacaat cttttcaaga ggtcttatcccaaaatcata aattcataat gatttatgaa 27480 caagaaaatg ttcatgtatt gttcttccaatcaacgatgg tgtcatatat gtcctctaaa 27540 atcactttcc ccagctagat cagtctatattaaaatgtct cacatagata gattgggtat 27600 ttttactcta atatataagg caacatgtgtcataaaatat aacactgctc aatattatta 27660 ttttaatata tgtgtatgag aaagaattc27689 2 148 PRT Arabidopsis thaliana 2 Met Pro Ile Gly Glu Leu Ala IleGly Ala Val Leu Gly Val Gly Ala 1 5 10 15 Gln Ala Ile Tyr Asp Arg PheArg Lys Ala Arg Asp Ile Ser Phe Val 20 25 30 His Arg Leu Cys Ala Thr IleLeu Ser Ile Glu Pro Phe Leu Val Gln 35 40 45 Ile Asp Lys Arg Ser Lys ValGlu Gly Ser Pro Leu Arg Glu Val Asn 50 55 60 Glu Arg Leu Thr Cys Phe LeuGlu Leu Ala Tyr Val Phe Val Glu Ala 65 70 75 80 Tyr Pro Lys Leu Arg ArgArg Gln Val Leu Arg Lys Tyr Arg Tyr Ile 85 90 95 Lys Ala Ile Glu Thr IleGlu Leu Ala Leu Arg Ser Ile Ile Val Val 100 105 110 Asp Phe Gln Val AspGln Trp Asp Asp Ile Lys Glu Ile Lys Ala Lys 115 120 125 Ile Ser Glu MetAsp Thr Lys Leu Ala Glu Val Ile Ser Ala Cys Ser 130 135 140 Lys Ile ArgAla 145 3 174 PRT Arabidopsis thaliana 3 Met Ile Ala Glu Val Ala Ala GlyGly Ala Leu Gly Leu Ala Leu Ser 1 5 10 15 Val Leu His Glu Ala Val LysArg Ala Lys Asp Arg Ser Val Thr Thr 20 25 30 Arg Phe Ile Leu His Arg LeuGlu Ala Thr Ile Asp Ser Ile Thr Pro 35 40 45 Leu Val Val Gln Ile Asp LysPhe Ser Glu Glu Met Glu Asp Ser Thr 50 55 60 Ser Arg Lys Val Asn Lys ArgLeu Lys Leu Leu Leu Glu Asn Ala Val 65 70 75 80 Ser Leu Val Glu Glu AsnAla Glu Leu Arg Arg Arg Asn Val Arg Lys 85 90 95 Lys Phe Arg Tyr Met ArgAsp Ile Lys Glu Phe Glu Ala Lys Leu Arg 100 105 110 Trp Val Val Asp ValAsp Val Gln Val Asn Gln Leu Ala Asp Ile Lys 115 120 125 Glu Leu Lys AlaLys Met Ser Glu Ile Ser Thr Lys Leu Asp Lys Ile 130 135 140 Met Pro GlnPro Lys Phe Glu Ile His Ile Gly Trp Cys Ser Gly Lys 145 150 155 160 ThrAsn Arg Ala Ile Arg Phe Thr Phe Cys Ser Asp Asp Ser 165 170 4 213 PRTArabidopsis thaliana 4 Met Pro Val Ser Glu Ile Met Ala Gly Ala Ala LeuGly Leu Ala Leu 1 5 10 15 Gln Val Leu His Asp Ala Ile Lys Lys Ala LysAsp Arg Ser Leu Thr 20 25 30 Thr Arg Cys Ile Leu Asp Arg Leu Asp Ala ThrIle Phe Arg Ile Thr 35 40 45 Pro Leu Val Thr Gln Val Asp Lys Leu Ser GluGlu Val Glu Asp Ser 50 55 60 Pro Arg Lys Val Ile Glu Asp Leu Lys His LeuLeu Glu Lys Ala Val 65 70 75 80 Ser Leu Val Glu Ala Tyr Ala Glu Leu ArgArg Arg Asn Leu Leu Lys 85 90 95 Lys Phe Arg Tyr Lys Arg Arg Ile Lys GluLeu Glu Ala Ser Leu Arg 100 105 110 Trp Met Val Asp Val Asp Val Gln ValAsn Gln Trp Val Asp Ile Lys 115 120 125 Glu Leu Met Ala Lys Met Ser GluMet Asn Thr Lys Leu Asp Glu Ile 130 135 140 Thr Arg Gln Pro Thr Asp CysIle Cys Phe Lys Ser Asn His Ser Thr 145 150 155 160 Ser Gln Ser Ser SerGln Asn Ile Val Glu Glu Thr Asp Arg Ser Leu 165 170 175 Glu Glu Ile ValGlu Cys Ser Ser Asp Gly Ser Lys Pro Lys Ile Asp 180 185 190 Ile His IleHis Trp Ser Ser Arg Lys Arg Asn Lys Asp Arg Glu Ile 195 200 205 Arg PheVal Leu Lys 210 5 205 PRT Arabidopsis thaliana 5 Met Pro Leu Thr Glu IleIle Ala Gly Ala Ala Leu Gly Leu Ala Leu 1 5 10 15 Gln Ile Leu His GluAla Ile Gln Arg Ala Lys Asp Arg Ser Leu Thr 20 25 30 Thr Ser Cys Ile LeuAsp Arg Leu Asp Ser Thr Ile Leu Arg Ile Thr 35 40 45 Pro Leu Met Ala LysVal Glu Lys Leu Asn Lys Glu Ser Asp Glu Ser 50 55 60 Leu Arg Lys Val PheGlu Asp Leu Lys His Leu Leu Glu Lys Ala Val 65 70 75 80 Val Leu Val GluAla Tyr Ala Glu Leu Lys Arg Arg Asn Leu Leu Glu 85 90 95 Lys Tyr Arg TyrLys Arg Arg Ile Lys Glu Leu Glu Gly Ser Leu Lys 100 105 110 Trp Met ValAsp Val Asp Val Lys Val Asn Gln Trp Ala Asp Ile Lys 115 120 125 Asp LeuMet Ala Lys Met Ser Glu Met Asn Thr Lys Leu Glu Lys Ile 130 135 140 MetGly Gln Pro Ile Asp Cys Ile Ile Ser Glu Asp Asn Thr Asn Met 145 150 155160 Asp Ile Val Glu Arg Val Asp Pro Ser Leu Glu Ala Lys Ala Gly Cys 165170 175 Ser Asn Ser Asp Ser Lys Pro Lys Ile Asp Ile His Leu Arg Trp Ser180 185 190 Lys Gln Ser Lys Asp His Gly Ile Arg Phe Val Leu Asn 195 200205 6 189 PRT Arabidopsis thaliana 6 Met Pro Leu Val Glu Leu Leu Thr SerAla Ala Leu Gly Leu Ser Leu 1 5 10 15 Gln Leu Leu His Glu Ala Ile IleArg Ala Lys Glu Lys Thr Leu Ile 20 25 30 Thr Arg Cys Ile Leu Asp Arg LeuAsp Ala Thr Leu His Lys Ile Thr 35 40 45 Pro Phe Val Ile Lys Ile Asp ThrLeu Thr Glu Glu Val Asp Glu Pro 50 55 60 Phe Arg Lys Val Ile Glu Glu LeuLys Arg Leu Leu Glu Lys Ala Ile 65 70 75 80 Arg Leu Val Asp Ala Tyr AlaGlu Leu Lys Leu Arg Asn Leu Leu Arg 85 90 95 Lys Tyr Arg Tyr Lys Arg ArgIle Lys Glu Leu Asp Ser Ser Leu Arg 100 105 110 Trp Met Ile Asp Val AspVal Gln Val Asn Gln Trp Leu Asp Ile Lys 115 120 125 Lys Leu Met Gly LysMet Ser Glu Met Asn Thr Lys Leu Asp Asp Ile 130 135 140 Thr Arg Gln ProMet Asp Ile Ile Glu Ala Thr Gly Arg Ser Ser Glu 145 150 155 160 Glu AspGly Cys Thr Lys Pro Thr Ile Asp Ile His Phe Arg Trp Lys 165 170 175 AsnGln Thr Lys Glu His Glu Ile Arg Phe Ile Phe Lys 180 185 7 4666 DNAArabidopsis thaliana 7 catgaaacat agatctcaaa agaagcgaaa taaaaagattattgttaatt attattttga 60 taaaattaca catagattga gaaagagttt ttcaataattatggggaata agagagagag 120 agagagaaat agatttccga aattgattac aagaagaaataatttcaaca aagtctctgt 180 ttttttttat caagctctta ttttactaca agcagaaataacttcagcaa gtttagtgtc 240 catttcagat atcttggcct tgatttcttt gatatcgtcccattgatcaa cttgaaaatc 300 cacaactatt atgcttctta atgcaagttc tatcgtttcgattgctttga tgtacctaaa 360 gataaacaga acaaacataa tactcgtgtt atttttccacaacatgatag gttttgtacg 420 tttagtgttt ggagattatc gaaatcatgt aaaaaaaattgttacaaaga agaagatatt 480 tttctctaaa ccattaaact aagaaattag gcgatccaaaaaccaataga aattcatgtc 540 atatatacga acctgtactt cctgagtact tgtctgcgtctgagtttcgg ataagcctca 600 acaaaaacat aagctaattc aaggaaacac gtgagacgttcgttgacttc ccttaatggt 660 gaaccttcca ctttactccg cttatcgatt tgaaccaaaaacggctcgat actaaggatt 720 gtagcgcaga gacggtgtac gaaagatata tctcttgcttttctgaaccg gtcgtaaatg 780 gcttgggctc caactccaag aacagcccct atcgcaagctcaccaatcgg cattttttga 840 aagtagttgt ttagctctcg aggtgaatat agaggaatctatgtacatgg aaggatggaa 900 ccatattaaa tagttttatg tttaacaagt taacgagtggttttaattat atgaagacaa 960 ttcaagagat tgactcatag acttagtact gtacgggtcaacaactctct ctttttctag 1020 gtaagaggag atcgttggat ctatatgcaa gttgtcgtgagtattaaatt acgtagaata 1080 ttattgaatt acgtcgaaga agcgagagtc aatctcactctcaatggtta acttgtacat 1140 ttagaagaag gaaaaatcaa cgaagttggc tgagtaagaagtgaagaaga aaaacagtga 1200 agaaagccaa aaagcagaag aggaaaatgg tggtatcaactaaaaatatt tcaacaaagg 1260 aagttactac taaaaatatt tcaacaaaag aagttactactaaaaataaa tactttgcat 1320 gttgcagtat atatttaaaa tttagaaata attatatctattaaaaaatc attttgtaac 1380 agatgttcga ttatgatata tagaattatt ttgtagacgttttataaaat agtttaaaaa 1440 attatattga agatatgaga tgaaccacaa tacgtatttttatttttcgt attttcaaat 1500 aaactcttat tattatatga aatctgaatt agcccagaatattattagat ttggtttata 1560 atttaatctc aaaattttct tccaaactga aaacagaaaaaaaaaaaaaa aaaaaaagaa 1620 gaagaagaag aagaagttaa aaaccactaa tctgaaagatccactctaat ttgtataaat 1680 ttttcgtttt aagttcaaag atgggatcaa atcaaatgagaagaatcctt aaaaactttc 1740 atctttatgt aagaagcaaa agcaaattta gttaagcttttttctaagtt ctttatatct 1800 tctttcagca ttaattcatt atccacaact ttgttatactcattatcctt caaacttgat 1860 tgtattgagt ttgcttctcc gttgatccta atacgctaagttcaactctt tgtaacaact 1920 ttgttcttta aagcattttg agttctaaat aaacaaattgagagaccaat gtggcagata 1980 atcgtcattt tgagatcgtt tgttgttttt tactctacaaactttggatt cacatacata 2040 tatatatata tatatataga tatatatata tatatatattgtaatgtaat gtatagtata 2100 tttctgaatt tctctttgtt taataaccat tggcacatttatttattttc aaagtatgtc 2160 attagattat tcatattaat acatatatat gagtcgtttgacacaattgg gacatcaaga 2220 atcatcactg cagaacgtaa atcggatcgc acggtttgtttttcctgaac accagccgat 2280 gtggatttca aacttcggtt gaggcattat tttgtcaagtttagtgctga tttcagacat 2340 cttggccttg agttctttga tatcagccaa ttgattaacttgaacatcca catctaccac 2400 ccatcgtaat ttagcttcga actctttgat atctctcatgtacctaaaga taaacaacac 2460 aaatataata cacatgttat tgacttaatt catagtaaatgttaggtttt gatagattta 2520 gtactgttgg gagtttatgg aaatcacata taggaactatttagcacaaa cctgaacttc 2580 ttgcgtacgt ttctgcgtct cagctccgca ttctcctcaacaagagaaac agcgttctca 2640 aggagaagct taagacgttt attgactttc ctcgatgttgaatcttccat ttcttcactg 2700 aacttatcaa tttgaaccac caacggtgtg atactatcgattgtagcttc gagacggtgt 2760 aagatgaatc ttgtggttac agatctatct tttgctcttttgacggcctc gtggaggaca 2820 ctgagagcaa gtccaagagc accccctgcg gcaacctcagcaatcatttt cttgaaatta 2880 gtttgttagc tctcgaggtg aagagttttt gatgagttatattgatgata ttattttgtt 2940 tggtaagaaa aatataagac catctattat attatatagaggtgaatatt tataattcct 3000 ttttcttctc aaatatttgg taaagtgttg ctctattaattcacataatg ttagtattat 3060 acacaaatat tataagggtg aatgcaatga gaaatctatgaacatggaag tcttttgctt 3120 aacaattaag ccgtgtagtt tgtataaagt caaacggatgttctttgttt ccgtaacttc 3180 ctacgaaaga gtgtgaataa gagatgtgtg gaccgcttggtaaagtacca tgcagttaga 3240 agcatgtacg gggtagtgaa acgtcgattt ttattataaaataaaataat aaacgatatg 3300 tgttggaggc gtatatatat taataaatag ttaaataacaaaattaaatc gtcttttact 3360 ttttttatag ctaataaaat caaatagttt aaagtcaattttagatcatt gtcagtaaaa 3420 acatcattaa actcaagtct ttcaaagtta atttaattaaatttatgcag aaaattcata 3480 aaacatagat ctcaaaagaa gcaaaataaa aagattattgttaattatta ttttgataaa 3540 attacacata gattgagaaa gagtttttca atcattattgggaagaaggg aggaagaaaa 3600 gaaaaaacag atttctgaaa ttgattataa gaagaaataatttcaacagt ctctgttttt 3660 ttaaatcaag ttcttatttt attacaaagt gaaataatttcagatatctt ggccttgatt 3720 tctttggtat ctttctaaaa aacaaattta gagaccaatgtggcagagaa tcgtcatttt 3780 gggatcgttt gttgtttttt actctacaaa ctttggattcacatacatat tatatgtatt 3840 gtaatgtaat gagtaatata tttctgaatg tctctttgtttacgttacat tggcacattt 3900 atgaagacaa aagacgtttt tgattaatta tattgatgatatatataaag acaaaagacg 3960 tttcacaaaa tattaaaacc ttaggaaaga caccccatttatcatcaatg gaggtgctct 4020 tagataacaa tctagaatcc ttatcgcttt agacagctgtgttattgact agtcatcatc 4080 taaagaggat aaggattgga aacgatttga attggagaccaagtgcttgg agagtaagct 4140 tagggttgtc tttgtatgtg tgtatatata ctcctcaagatcgatcaata acatcaagca 4200 ctttttcaac cattcttagt ctttacaatt aatgtacgaagaggattatt atttattaaa 4260 ttacgaaaaa gaagtgaaaa tcgatctaaa tgattgactttttacgtaga atcgtcgaat 4320 tgcatgtaca tttccaccga aattccaaaa atctgaattacaaataagtt ggaaccgatc 4380 gatcttgttt tgtatattta cgtacaaggc agacgtacatacatgtagtt tggattatca 4440 tatgtatgat caacgcaatt ttcgtgaata gaaacgtgaatactaacaat ttcggtgaat 4500 acctaccgta aatactaaca ttaaaatcta tgacttcttaaaataataat caatcaaact 4560 tttacatttg attttatatt ttcctcagtt tttaggcctatgatacacct gccttctcaa 4620 aatattagtt ccgtgatgtt tgctccatct aaggtggatatcgatc 4666 8 447 DNA Artificial Sequence Description of ArtificialSequence cDNA from A. thaliana 8 atgccgattg gtgagcttgc gataggggctgttcttggag ttggagccca agccatttac 60 gaccggttca gaaaagcaag agatatatctttcgtacacc gtctctgcgc tacaatcctt 120 agtatcgagc cgtttttggt tcaaatcgataagcggagta aagtggaagg ttcaccatta 180 agggaagtca acgaacgtct cacgtgtttccttgaattag cttatgtttt tgttgaggct 240 tatccgaaac tcagacgcag acaagtactcaggaagtaca ggtacatcaa agcaatcgaa 300 acgatagaac ttgcattaag aagcataatagttgtggatt ttcaagttga tcaatgggac 360 gatatcaaag aaatcaaggc caagatatctgaaatggaca ctaaacttgc tgaagttatt 420 tctgcttgta gtaaaataag agcttga 447 9447 DNA Artificial Sequence Description of Artificial Sequence cDNA fromA. thaliana 9 atgccgattg gtgagcttgc gataggggct gttcttggag ttggagcccaagccatttac 60 gaccgcttca gaaaagcaag agatatatct ttcgtacacc gtctctgcgctacaatcctt 120 agtatcgagc cgtttttggt tcaaatcgat aagcggagta aagtggaaggttcaccatta 180 agggaagtca acgaacgtct cacgtgtttc cttgaattag cttatgtttttgttgaggct 240 tatccgaaac tcagacgcag acaagtactc aggaagtaca ggtacatcaaagcaatcgaa 300 acgatagaac ttgcattaag aagcataata gttgtggatt ttcaagttgatcaatgggac 360 gatatcaaag aaatcaaggc caagatatct gaaatggaca ctaaacttgctgaagttatt 420 tctgcttgta gtaaaataag agcttga 447 10 510 DNA ArtificialSequence Description of Artificial Sequence cDNA from A. thaliana 10atgccgattg gtgagcttgc gataggggct gttcttggag ttggagccca agccatttac 60gaccgcttca gaaaagcaag agatatatct ttcgtacacc gtctctgcgc tacaatcatt 120agtatcgagc cgtttttggt tcaaatcgat aagcggagta aagtggaagg ttcaccatta 180agggaagtta acgaacgtct cacgtgtttc cttgaattag cttatgtttt agttgaggct 240tatccgaaac tcagacgcag acaagtactc aggaagtaca ggtgcatcaa agcaatcgaa 300acgatagaac ttgcattaag aaggataata gttgtggatt ttcaagttga tcaatgggac 360gatatcaaag aaatcaaggc caagatatct gaaacggaca ctaaacttgc tgatcaatgg 420gacgatatca aagaaatcaa ggccaagata tctgaaatgg acactaaact tgctgaagtt 480atttctgctt gtagtaaaat aagaacttga 510 11 447 DNA Artificial SequenceDescription of Artificial Sequence cDNA from A. thaliana 11 atgccgattggtgagcttgc gataggggct gttcttggag ttggagccca agccatttac 60 gaccggttcagaaaagcaag agatatatct ttcgtacacc gtctctgcgc tacaatcctt 120 agtatcgagccgttgttggt tcaaatcgat aagcggagta aagtggaagg ttcaccatta 180 agggaagtcaacgaacgtct cacgtgtttc cttgaattag cttatgtttt agttgaggct 240 tatccgaaactcagacgcag acaagtactc aggaagtaca ggtacatcaa agcaatcgaa 300 acgatagaacttgcattaag aagcataata gttgtggatt ttcaagttga tcaatgggac 360 gatatcaaagaaatcaaggc caagatatct gaaatggaca ctaaacttgc tgaagttatt 420 tctgcttgtagtaaaataag agcttga 447 12 447 DNA Artificial Sequence Description ofArtificial Sequence cDNA from A. thaliana 12 atgccgattg gtgagcttgcgataggggct gttcttggag ttggagccca agccatttac 60 gaccgcttca gaaaagcaagagatatatct gtcgtaaacc gtctctgcgc tacaatcatt 120 agtatcaggc cgttgttggttcaaatcgat aagcggagta aagtggaagg ttcaccatta 180 agggaagtca acgaacgtctcacgtgtttc cttgaattag cttatgtttt agttgaggct 240 tatccgaaac tcagacgcagacaagtactc aggaagtaca ggtacatcaa agcaatcgaa 300 acgatagaac ttgcattaagaagcataata gttgtggatt ttcaagttga tcaatgggac 360 gatatcaaag aaatcaaggccaagatatct gaaatggaca ctaaacttgc tgaagttatt 420 tctgcttgta gtaaaataagagcttga 447 13 447 DNA Artificial Sequence Description of ArtificialSequence cDNA from A. thaliana 13 atgccgattg gtgagcttgc gataggggctgttcttggag ttggagccca agccatttac 60 gaccgcttca gaaaagcaag agatatatctgtcgtaaacc gtctctgcgc tacaatcatt 120 agtatcaggc cgttgttggt tcaaatcgataagcggagta aagtggaagg ttcaccatta 180 agggaagtca acgaacgtct cacgtgtttccttgaattag cttatgtttt tgttgaggct 240 tatccgaaac tcagacgcag acaagtactcaggaagtaca ggtacatcaa agcaatcgaa 300 acgatagaac ttgcattaag aagcataatagttgtggatt ttcaagttga tcaatgggac 360 gatatcaaag aaatcaaggc caagatatctgaaatggaca ctaaacttgc tgaagttatt 420 tctgcttgta gtaaaataag aacttga 44714 148 PRT Arabidopsis thaliana 14 Met Pro Ile Gly Glu Leu Ala Ile GlyAla Val Leu Gly Val Gly Ala 1 5 10 15 Gln Ala Ile Tyr Asp Arg Phe ArgLys Ala Arg Asp Ile Ser Phe Val 20 25 30 His Arg Leu Cys Ala Thr Ile LeuSer Ile Glu Pro Phe Leu Val Gln 35 40 45 Ile Asp Lys Arg Ser Lys Val GluGly Ser Pro Leu Arg Glu Val Asn 50 55 60 Glu Arg Leu Thr Cys Phe Leu GluLeu Ala Tyr Val Phe Val Glu Ala 65 70 75 80 Tyr Pro Lys Leu Arg Arg ArgGln Val Leu Arg Lys Tyr Arg Tyr Ile 85 90 95 Lys Ala Ile Glu Thr Ile GluLeu Ala Leu Arg Ser Ile Ile Val Val 100 105 110 Asp Phe Gln Val Asp GlnTrp Asp Asp Ile Lys Glu Ile Lys Ala Lys 115 120 125 Ile Ser Glu Met AspThr Lys Leu Ala Glu Val Ile Ser Ala Cys Ser 130 135 140 Lys Ile Arg Ala145 15 169 PRT Arabidopsis thaliana 15 Met Pro Ile Gly Glu Leu Ala IleGly Ala Val Leu Gly Val Gly Ala 1 5 10 15 Gln Ala Ile Tyr Asp Arg PheArg Lys Ala Arg Asp Ile Ser Phe Val 20 25 30 His Arg Leu Cys Ala Thr IleIle Ser Ile Glu Pro Phe Leu Val Gln 35 40 45 Ile Asp Lys Arg Ser Lys ValGlu Gly Ser Pro Leu Arg Glu Val Asn 50 55 60 Glu Arg Leu Thr Cys Phe LeuGlu Leu Ala Tyr Val Leu Val Glu Ala 65 70 75 80 Tyr Pro Lys Leu Arg ArgArg Gln Val Leu Arg Lys Tyr Arg Cys Ile 85 90 95 Lys Ala Ile Glu Thr IleGlu Leu Ala Leu Arg Arg Ile Ile Val Val 100 105 110 Asp Phe Gln Val AspGln Trp Asp Asp Ile Lys Glu Ile Lys Ala Lys 115 120 125 Ile Ser Glu ThrAsp Thr Lys Leu Ala Asp Gln Trp Asp Asp Ile Lys 130 135 140 Glu Ile LysAla Lys Ile Ser Glu Met Asp Thr Lys Leu Ala Glu Val 145 150 155 160 IleSer Ala Cys Ser Lys Ile Arg Thr 165 16 148 PRT Arabidopsis thaliana 16Met Pro Ile Gly Glu Leu Ala Ile Gly Ala Val Leu Gly Val Gly Ala 1 5 1015 Gln Ala Ile Tyr Asp Arg Phe Arg Lys Ala Arg Asp Ile Ser Phe Val 20 2530 His Arg Leu Cys Ala Thr Ile Leu Ser Ile Glu Pro Leu Leu Val Gln 35 4045 Ile Asp Lys Arg Ser Lys Val Glu Gly Ser Pro Leu Arg Glu Val Asn 50 5560 Glu Arg Leu Thr Cys Phe Leu Glu Leu Ala Tyr Val Leu Val Glu Ala 65 7075 80 Tyr Pro Lys Leu Arg Arg Arg Gln Val Leu Arg Lys Tyr Arg Tyr Ile 8590 95 Lys Ala Ile Glu Thr Ile Glu Leu Ala Leu Arg Ser Ile Ile Val Val100 105 110 Asp Phe Gln Val Asp Gln Trp Asp Asp Ile Lys Glu Ile Lys AlaLys 115 120 125 Ile Ser Glu Met Asp Thr Lys Leu Ala Glu Val Ile Ser AlaCys Ser 130 135 140 Lys Ile Arg Ala 145 17 148 PRT Arabidopsis thaliana17 Met Pro Ile Gly Glu Leu Ala Ile Gly Ala Val Leu Gly Val Gly Ala 1 510 15 Gln Ala Ile Tyr Asp Arg Phe Arg Lys Ala Arg Asp Ile Ser Val Val 2025 30 Asn Arg Leu Cys Ala Thr Ile Ile Ser Ile Arg Pro Leu Leu Val Gln 3540 45 Ile Asp Lys Arg Ser Lys Val Glu Gly Ser Pro Leu Arg Glu Val Asn 5055 60 Glu Arg Leu Thr Cys Phe Leu Glu Leu Ala Tyr Val Leu Val Glu Ala 6570 75 80 Tyr Pro Lys Leu Arg Arg Arg Gln Val Leu Arg Lys Tyr Arg Tyr Ile85 90 95 Lys Ala Ile Glu Thr Ile Glu Leu Ala Leu Arg Ser Ile Ile Val Val100 105 110 Asp Phe Gln Val Asp Gln Trp Asp Asp Ile Lys Glu Ile Lys AlaLys 115 120 125 Ile Ser Glu Met Asp Thr Lys Leu Ala Glu Val Ile Ser AlaCys Ser 130 135 140 Lys Ile Arg Ala 145 18 148 PRT Arabidopsis thaliana18 Met Pro Ile Gly Glu Leu Ala Ile Gly Ala Val Leu Gly Val Gly Ala 1 510 15 Gln Ala Ile Tyr Asp Arg Phe Arg Lys Ala Arg Asp Ile Ser Val Val 2025 30 Asn Arg Leu Cys Ala Thr Ile Ile Ser Ile Arg Pro Leu Leu Val Gln 3540 45 Ile Asp Lys Arg Ser Lys Val Glu Gly Ser Pro Leu Arg Glu Val Asn 5055 60 Glu Arg Leu Thr Cys Phe Leu Glu Leu Ala Tyr Val Phe Val Glu Ala 6570 75 80 Tyr Pro Lys Leu Arg Arg Arg Gln Val Leu Arg Lys Tyr Arg Tyr Ile85 90 95 Lys Ala Ile Glu Thr Ile Glu Leu Ala Leu Arg Ser Ile Ile Val Val100 105 110 Asp Phe Gln Val Asp Gln Trp Asp Asp Ile Lys Glu Ile Lys AlaLys 115 120 125 Ile Ser Glu Met Asp Thr Lys Leu Ala Glu Val Ile Ser AlaCys Ser 130 135 140 Lys Ile Arg Thr 145 19 525 DNA Artificial SequenceDescription of Artificial Sequence cDNA from A. thaliana 19 atgattgctgaggttgccgc agggggtgct cttggacttg ctctcagtgt cctccacgag 60 gccgtcaaaagagcaaaaga tagatctgta accacaagat tcatcttaca ccgtctcgaa 120 gctacaatcgatagtatcac accgttggtg gttcaaattg ataagttcag tgaagaaatg 180 gaagattcaacatcgaggaa agtcaataaa cgtcttaagc ttctccttga gaacgctgtt 240 tctcttgttgaggagaatgc ggagctgaga cgcagaaacg tacgcaagaa gttcaggtac 300 atgagagatatcaaagagtt cgaagctaaa ttacgatggg tggtagatgt ggatgttcaa 360 gttaatcaattggctgatat caaagaactc aaggccaaga tgtctgaaat cagcactaaa 420 cttgacaaaataatgcctca accgaagttt gaaatccaca tcggctggtg ttcaggaaaa 480 acaaaccgtgcgatccgatt tacgttctgc agtgatgatt cttga 525 20 525 DNA ArtificialSequence Description of Artificial Sequence cDNA from A. thaliana 20atgattgctg aggttgcggc agggggtgct cttggacttg ctctcagtgt ccttcaagag 60gccgtcaaaa gagcaaaaga tagatctgta accacaagat tcatcttaca ccgtctcgaa 120gctacaatcg atagtatcac tccgttggtg gttcaaattg ataagttcag tgaagaaatg 180gaagattcat catcgaggaa agtcaataaa cgtcttaagc ttctccttga gaacgctgtt 240tctcttgttg aggagaatgc ggagctgaga cgcagaaacg tacgcaagaa gttcaggtac 300atgagagata tcaaagagtt cgaagctaag atacgatggg tggtaggtgt ggatgttcaa 360gttaatcaat tggctgatat caaagaactc aaggccaaga tgtctgaaat cagcactaaa 420cttgacaaaa taatgcctca accgaagttt gaaatccaca tcggctggtg ttcaggaaaa 480aaaaaccgtg cgatccgatt tacgttctgc agtgatgatt cttga 525 21 525 DNAArtificial Sequence Description of Artificial Sequence cDNA from A.thaliana 21 atgattgctg aggttgccgc agggggtgct cttggacttg ctctcagtgtcctccacgag 60 gccgtcaaaa gagcaaaaga tagatctgta accacaagat tcatcttacaccgtctcgaa 120 gctacaatcg atagtatcac accgttggtg gttcaaattg ataagttcagtgaagaaatg 180 gaagattcat catcgaggaa agtcaataaa cgtcttaagc ttctccttgagaacgctgtt 240 tctcttgttg aggagaatgc ggagctgaga cgcagaaacg tacgcaagaagttcaggtac 300 atgagagata tcaaagagtt cgaagctaaa ttacgatggg tggtaggtgtggatgttcaa 360 gttaatcaat tggctgatat caaagaactc aaggccaaga tgtctgaaatcagcactaaa 420 cttgacaaaa taatgcctca accgaagttt gaaatccaca tcggctggtgttcaggaaaa 480 acaaaccgtg cgatccgatt tacgttctgc agtgatgatt cttga 525 22525 DNA Artificial Sequence Description of Artificial Sequence cDNA fromA. thaliana 22 atgattgctg aggttgcggc agggggtgct cttggacttg ctctcagtgtcctccacgag 60 gccgtcaaaa gagcaaaaga tagatctgta accacaagat tcatcttacaccgtctcgaa 120 gctacaatcg atagtatcac tccgttggtg gttcaaattg ataagttcagtgaagaaatg 180 gaagattcat catcgaggaa agtcaataaa cgtcttaagc ttctccttgagaacgctgtt 240 tctcttgttg aggagaatgc ggagctgaga cgcagaaacg tacgcaagaagttcaggtac 300 atgagagata tcaaagagtt cgaagctaaa ttacgatggg tggtaggtgtggatgttcaa 360 gttaatcaat tggctgatat caaagaactc aaggccaaga tgtctgaaatcagcactaaa 420 cttgacaaaa taatgcctca accgaagttt gaaatccaca tcggctggtgttcaggaaaa 480 aaaaaccgtg cgatccgatt tacgttctgc agtgatgatt cttga 525 23524 DNA Artificial Sequence Description of Artificial Sequence cDNA fromA. thaliana 23 atgattgctg aggttgccgc agggggtgct cttggacttg ctctcagtttcctccacgag 60 gccgtcaaaa gagcaaaaga tagatctgta accacaagat tcatcttacaccgtctcgaa 120 gctacaatcg atagtatcac tccgttggtg gttcaaattg ataagttcagtgaagaaatg 180 gaagattcat catcgaggaa agtcaataaa cgtcttaagc ttctccttgagaacgctgtt 240 tctcttgttg aggagaatgc ggagctgaga cgcagaaacg tacgcaagaagttcaggtac 300 atgagagata tcaaagagtt cgaagctaaa ttacgatggg tggtaggtgtggatgttcaa 360 gttaatcaat tggctgatat caaagaactc aaggccaaga tgtctgaaatcagcactaaa 420 cttgacaaat aatgcctcaa ccgaagtttg aaatccacat cggctggtgttcaggaaaaa 480 aaaaccgtgc gatccgattt acgttctgca gtgatgattc ttga 524 24525 DNA Artificial Sequence Description of Artificial Sequence cDNA fromA. thaliana 24 atgattgctg aggttgccgc agggggtgct cttggacttg ctctcagtgtcctccacgag 60 gccgtcaaaa gagcaaaaga tagatctgta accacaagat tcatcttacaccgtctcgaa 120 gctacaatcg atagtatcac tccgttggtg gttcaaattg ataagttcagtgaagaaatg 180 gaagattcat catcgaggaa agtcaatgaa cgtcttaagc ttctccttgagaacgctgtt 240 tctcttgttg aggagaatgc ggagctgaga cgcagaaacg tacgcaagaagttcaggtac 300 atgagagata tcaaagagtt cgaagctaaa ttacgatggg tggtaggtgtggatgttcaa 360 gttaatcaat tggctgatat caaagaactc aaggccaaga tgtctgaaatcagcactaaa 420 cttgacaaaa taatgcctca accgaagttt gaaatccaca tcggctggtgttcaggaaaa 480 acaaaccgtg cgatccgatt tacgttctgc agtgatgatt cttga 525 25174 PRT Arabidopsis thaliana 25 Met Ile Ala Glu Val Ala Ala Gly Gly AlaLeu Gly Leu Ala Leu Ser 1 5 10 15 Val Leu His Glu Ala Val Lys Arg AlaLys Asp Arg Ser Val Thr Thr 20 25 30 Arg Phe Ile Leu His Arg Leu Glu AlaThr Ile Asp Ser Ile Thr Pro 35 40 45 Leu Val Val Gln Ile Asp Lys Phe SerGlu Glu Met Glu Asp Ser Thr 50 55 60 Ser Arg Lys Val Asn Lys Arg Leu LysLeu Leu Leu Glu Asn Ala Val 65 70 75 80 Ser Leu Val Glu Glu Asn Ala GluLeu Arg Arg Arg Asn Val Arg Lys 85 90 95 Lys Phe Arg Tyr Met Arg Asp IleLys Glu Phe Glu Ala Lys Leu Arg 100 105 110 Trp Val Val Asp Val Asp ValGln Val Asn Gln Leu Ala Asp Ile Lys 115 120 125 Glu Leu Lys Ala Lys MetSer Glu Ile Ser Thr Lys Leu Asp Lys Ile 130 135 140 Met Pro Gln Pro LysPhe Glu Ile His Ile Gly Trp Cys Ser Gly Lys 145 150 155 160 Thr Asn ArgAla Ile Arg Phe Thr Phe Cys Ser Asp Asp Ser 165 170 26 174 PRTArabidopsis thaliana 26 Met Ile Ala Glu Val Ala Ala Gly Gly Ala Leu GlyLeu Ala Leu Ser 1 5 10 15 Val Leu Gln Glu Ala Val Lys Arg Ala Lys AspArg Ser Val Thr Thr 20 25 30 Arg Phe Ile Leu His Arg Leu Glu Ala Thr IleAsp Ser Ile Thr Pro 35 40 45 Leu Val Val Gln Ile Asp Lys Phe Ser Glu GluMet Glu Asp Ser Ser 50 55 60 Ser Arg Lys Val Asn Lys Arg Leu Lys Leu LeuLeu Glu Asn Ala Val 65 70 75 80 Ser Leu Val Glu Glu Asn Ala Glu Leu ArgArg Arg Asn Val Arg Lys 85 90 95 Lys Phe Arg Tyr Met Arg Asp Ile Lys GluPhe Glu Ala Lys Ile Arg 100 105 110 Trp Val Val Gly Val Asp Val Gln ValAsn Gln Leu Ala Asp Ile Lys 115 120 125 Glu Leu Lys Ala Lys Met Ser GluIle Ser Thr Lys Leu Asp Lys Ile 130 135 140 Met Pro Gln Pro Lys Phe GluIle His Ile Gly Trp Cys Ser Gly Lys 145 150 155 160 Lys Asn Arg Ala IleArg Phe Thr Phe Cys Ser Asp Asp Ser 165 170 27 174 PRT Arabidopsisthaliana 27 Met Ile Ala Glu Val Ala Ala Gly Gly Ala Leu Gly Leu Ala LeuSer 1 5 10 15 Val Leu His Glu Ala Val Lys Arg Ala Lys Asp Arg Ser ValThr Thr 20 25 30 Arg Phe Ile Leu His Arg Leu Glu Ala Thr Ile Asp Ser IleThr Pro 35 40 45 Leu Val Val Gln Ile Asp Lys Phe Ser Glu Glu Met Glu AspSer Ser 50 55 60 Ser Arg Lys Val Asn Lys Arg Leu Lys Leu Leu Leu Glu AsnAla Val 65 70 75 80 Ser Leu Val Glu Glu Asn Ala Glu Leu Arg Arg Arg AsnVal Arg Lys 85 90 95 Lys Phe Arg Tyr Met Arg Asp Ile Lys Glu Phe Glu AlaLys Leu Arg 100 105 110 Trp Val Val Gly Val Asp Val Gln Val Asn Gln LeuAla Asp Ile Lys 115 120 125 Glu Leu Lys Ala Lys Met Ser Glu Ile Ser ThrLys Leu Asp Lys Ile 130 135 140 Met Pro Gln Pro Lys Phe Glu Ile His IleGly Trp Cys Ser Gly Lys 145 150 155 160 Thr Asn Arg Ala Ile Arg Phe ThrPhe Cys Ser Asp Asp Ser 165 170 28 174 PRT Arabidopsis thaliana 28 MetIle Ala Glu Val Ala Ala Gly Gly Ala Leu Gly Leu Ala Leu Ser 1 5 10 15Val Leu His Glu Ala Val Lys Arg Ala Lys Asp Arg Ser Val Thr Thr 20 25 30Arg Phe Ile Leu His Arg Leu Glu Ala Thr Ile Asp Ser Ile Thr Pro 35 40 45Leu Val Val Gln Ile Asp Lys Phe Ser Glu Glu Met Glu Asp Ser Ser 50 55 60Ser Arg Lys Val Asn Lys Arg Leu Lys Leu Leu Leu Glu Asn Ala Val 65 70 7580 Ser Leu Val Glu Glu Asn Ala Glu Leu Arg Arg Arg Asn Val Arg Lys 85 9095 Lys Phe Arg Tyr Met Arg Asp Ile Lys Glu Phe Glu Ala Lys Leu Arg 100105 110 Trp Val Val Gly Val Asp Val Gln Val Asn Gln Leu Ala Asp Ile Lys115 120 125 Glu Leu Lys Ala Lys Met Ser Glu Ile Ser Thr Lys Leu Asp LysIle 130 135 140 Met Pro Gln Pro Lys Phe Glu Ile His Ile Gly Trp Cys SerGly Lys 145 150 155 160 Lys Asn Arg Ala Ile Arg Phe Thr Phe Cys Ser AspAsp Ser 165 170 29 143 PRT Arabidopsis thaliana 29 Met Ile Ala Glu ValAla Ala Gly Gly Ala Leu Gly Leu Ala Leu Ser 1 5 10 15 Phe Leu His GluAla Val Lys Arg Ala Lys Asp Arg Ser Val Thr Thr 20 25 30 Arg Phe Ile LeuHis Arg Leu Glu Ala Thr Ile Asp Ser Ile Thr Pro 35 40 45 Leu Val Val GlnIle Asp Lys Phe Ser Glu Glu Met Glu Asp Ser Ser 50 55 60 Ser Arg Lys ValAsn Lys Arg Leu Lys Leu Leu Leu Glu Asn Ala Val 65 70 75 80 Ser Leu ValGlu Glu Asn Ala Glu Leu Arg Arg Arg Asn Val Arg Lys 85 90 95 Lys Phe ArgTyr Met Arg Asp Ile Lys Glu Phe Glu Ala Lys Leu Arg 100 105 110 Trp ValVal Gly Val Asp Val Gln Val Asn Gln Leu Ala Asp Ile Lys 115 120 125 GluLeu Lys Ala Lys Met Ser Glu Ile Ser Thr Lys Leu Asp Lys 130 135 140 30174 PRT Arabidopsis thaliana 30 Met Ile Ala Glu Val Ala Ala Gly Gly AlaLeu Gly Leu Ala Leu Ser 1 5 10 15 Val Leu His Glu Ala Val Lys Arg AlaLys Asp Arg Ser Val Thr Thr 20 25 30 Arg Phe Ile Leu His Arg Leu Glu AlaThr Ile Asp Ser Ile Thr Pro 35 40 45 Leu Val Val Gln Ile Asp Lys Phe SerGlu Glu Met Glu Asp Ser Ser 50 55 60 Ser Arg Lys Val Asn Glu Arg Leu LysLeu Leu Leu Glu Asn Ala Val 65 70 75 80 Ser Leu Val Glu Glu Asn Ala GluLeu Arg Arg Arg Asn Val Arg Lys 85 90 95 Lys Phe Arg Tyr Met Arg Asp IleLys Glu Phe Glu Ala Lys Leu Arg 100 105 110 Trp Val Val Gly Val Asp ValGln Val Asn Gln Leu Ala Asp Ile Lys 115 120 125 Glu Leu Lys Ala Lys MetSer Glu Ile Ser Thr Lys Leu Asp Lys Ile 130 135 140 Met Pro Gln Pro LysPhe Glu Ile His Ile Gly Trp Cys Ser Gly Lys 145 150 155 160 Thr Asn ArgAla Ile Arg Phe Thr Phe Cys Ser Asp Asp Ser 165 170 31 756 DNA Brassicarapa 31 atgcctattg gtgaagttat tgtaggggct gctcttggaa ttactctgcaagtgcttcat 60 gaagctatca taaaagcaaa agatagatct tcaaccaaaa aaagtatcttggaccgcctc 120 gatgctacaa tctccaggat cactccgttg gtggttcatg tcgataagatcagcaaaaga 180 gtagaagatt ctgagaggaa agtcattgaa gaactcaagc gtcttcttgaaaaggctgtt 240 tctcttgttg aggcttatgc agaactcaga cgcagaaacc tacacaagaagcataggttt 300 gtatagttta tataatacat gaaatacttg aaaaagtctt tgtgatttcttaaaatgttt 360 ttatttggtt tacataatat ttatgtgttg ttgatatata ggtgcaagagtagaatcaaa 420 gagttagaag tttcattaag atggatgata gatgtggatg ttcaagtcaaccaatggcta 480 gatatcaaaa aactcgtggt taagatgtct gaaatgaaca caaaactcgacaagatcacg 540 tgccaaccaa ctgatggtag ttgtttcaag agcaatgata gcacatcaccagtgttttca 600 caaagtagta gtagtctcga agcaacagac ggatcttcag aggaagatgaagaagaaagc 660 ccaagtaatg gatctgaacc aaggatcgat atccacctgc gatggagttcaagaaaagga 720 agaaaagatc gtgagatccg attcatggcc aagtga 756 32 651 DNAArtificial Sequence Description of Artificial Sequence cDNA from B. rapa32 atgcctattg gtgaagttat tgtaggggct gctcttggaa ttactctgca agtgcttcat 60gaagctatca taaaagcaaa agatagatct tcaaccaaaa aaagtatctt ggaccgcctc 120gatgctacaa tctccaggat cactccgttg gtggttcatg tcgataagat cagcaaaaga 180gtagaagatt ctgagaggaa agtcattgaa gaactcaagc gtcttcttga aaaggctgtt 240tctcttgttg aggcttatgc agaactcaga cgcagaaacc tacacaagaa gcataggtgc 300aagagtagaa tcaaagagtt agaagtttca ttaagatgga tgatagatgt ggatgttcaa 360gtcaaccaat ggctagatat caaaaaactc gtggttaaga tgtctgaaat gaacacaaaa 420ctcgacaaga tcacgtgcca accaactgat ggtagttgtt tcaagagcaa tgatagcaca 480tcaccagtgt tttcacaaag tagtagtagt ctcgaagcaa cagacggatc ttcagaggaa 540gatgaagaag aaagcccaag taatggatct gaaccaagga tcgatatcca cctgcgatgg 600agttcaagaa aaggaagaaa agatcgtgag atccgattca tggccaagtg a 651 33 216 PRTBrassica rapa 33 Met Pro Ile Gly Glu Val Ile Val Gly Ala Ala Leu Gly IleThr Leu 1 5 10 15 Gln Val Leu His Glu Ala Ile Ile Lys Ala Lys Asp ArgSer Ser Thr 20 25 30 Lys Lys Ser Ile Leu Asp Arg Leu Asp Ala Thr Ile SerArg Ile Thr 35 40 45 Pro Leu Val Val His Val Asp Lys Ile Ser Lys Arg ValGlu Asp Ser 50 55 60 Glu Arg Lys Val Ile Glu Glu Leu Lys Arg Leu Leu GluLys Ala Val 65 70 75 80 Ser Leu Val Glu Ala Tyr Ala Glu Leu Arg Arg ArgAsn Leu His Lys 85 90 95 Lys His Arg Cys Lys Ser Arg Ile Lys Glu Leu GluVal Ser Leu Arg 100 105 110 Trp Met Ile Asp Val Asp Val Gln Val Asn GlnTrp Leu Asp Ile Lys 115 120 125 Lys Leu Val Val Lys Met Ser Glu Met AsnThr Lys Leu Asp Lys Ile 130 135 140 Thr Cys Gln Pro Thr Asp Gly Ser CysPhe Lys Ser Asn Asp Ser Thr 145 150 155 160 Ser Pro Val Phe Ser Gln SerSer Ser Ser Leu Glu Ala Thr Asp Gly 165 170 175 Ser Ser Glu Glu Asp GluGlu Glu Ser Pro Ser Asn Gly Ser Glu Pro 180 185 190 Arg Ile Asp Ile HisLeu Arg Trp Ser Ser Arg Lys Gly Arg Lys Asp 195 200 205 Arg Glu Ile ArgPhe Met Ala Lys 210 215 34 753 DNA Brassica rapa 34 atgcctattggtgaggttat tgtaggggct gctcttggaa ttactctgca agtgcttcat 60 caagctatcataaaagcaaa agatagatct tcaaccacaa aatgtatctt ggtccgcctc 120 gatgctacaatctccaggat cactccgttg gtggttcatg tcgataagat cagcaaaaga 180 gtagaagattctgagaggaa agtcattgaa gaactcaagc gtcttcttga aaaggctgtt 240 tctcttgttgaggcttatgc agaactcaga cgcagaaacc tacacaagaa gcattggttt 300 gtatagtttatataatacat gaaatacttg aaaaagtctt tgtgatttct taaaatgttt 360 ttatttggtttacataatat ttatgtgttg ttgatatata ggtacaagag tagaatcaaa 420 gagttagaagcttcattaag atggatggta gatgtggatg ttcaagtcaa ccaatggcta 480 gatatcaaagaactcgtggc taagatgtct gaaatgaaca caaaactcga caagatcacg 540 agccaaccaactgatggtag ttgtttcaag agcaatgata gcatatcacc agtgttatca 600 caaagtagtaggatcgaagc aacagacgga tcttcagagg aagatgaaga agaaagctca 660 agtaatggatccgaaccaag gatcgatatc cacctgcgat ggagttcaag aaaaggaaga 720 aaagatcgtgagatccgatt cacggccaag tga 753 35 648 DNA Artificial Sequence Descriptionof Artificial Sequence cDNA from B. rapa 35 atgcctattg gtgaggttattgtaggggct gctcttggaa ttactctgca agtgcttcat 60 caagctatca taaaagcaaaagatagatct tcaaccacaa aatgtatctt ggtccgcctc 120 gatgctacaa tctccaggatcactccgttg gtggttcatg tcgataagat cagcaaaaga 180 gtagaagatt ctgagaggaaagtcattgaa gaactcaagc gtcttcttga aaaggctgtt 240 tctcttgttg aggcttatgcagaactcaga cgcagaaacc tacacaagaa gtataggtac 300 aagagtagaa tcaaagagttagaagcttca ttaagatgga tggtagatgt ggatgttcaa 360 gtcaaccaat ggctagatatcaaagaactc gtggctaaga tgtctgaaat gaacacaaaa 420 ctcgacaaga tcacgagccaaccaactgat ggtagttgtt tcaagagcaa tgatagcata 480 tcaccagtgt tatcacaaagtagtaggatc gaagcaacag acggatcttc agaggaagat 540 gaagaagaaa gctcaagtaatggatccgaa ccaaggatcg atatccacct gcgatggagt 600 tcaagaaaag gaagaaaagatcgtgagatc cgattcacgg ccaagtga 648 36 215 PRT Brassica rapa 36 Met ProIle Gly Glu Val Ile Val Gly Ala Ala Leu Gly Ile Thr Leu 1 5 10 15 GlnVal Leu His Gln Ala Ile Ile Lys Ala Lys Asp Arg Ser Ser Thr 20 25 30 ThrLys Cys Ile Leu Val Arg Leu Asp Ala Thr Ile Ser Arg Ile Thr 35 40 45 ProLeu Val Val His Val Asp Lys Ile Ser Lys Arg Val Glu Asp Ser 50 55 60 GluArg Lys Val Ile Glu Glu Leu Lys Arg Leu Leu Glu Lys Ala Val 65 70 75 80Ser Leu Val Glu Ala Tyr Ala Glu Leu Arg Arg Arg Asn Leu His Lys 85 90 95Lys Tyr Arg Tyr Lys Ser Arg Ile Lys Glu Leu Glu Ala Ser Leu Arg 100 105110 Trp Met Val Asp Val Asp Val Gln Val Asn Gln Trp Leu Asp Ile Lys 115120 125 Glu Leu Val Ala Lys Met Ser Glu Met Asn Thr Lys Leu Asp Lys Ile130 135 140 Thr Ser Gln Pro Thr Asp Gly Ser Cys Phe Lys Ser Asn Asp SerIle 145 150 155 160 Ser Pro Val Leu Ser Gln Ser Ser Arg Ile Glu Ala ThrAsp Gly Ser 165 170 175 Ser Glu Glu Asp Glu Glu Glu Ser Ser Ser Asn GlySer Glu Pro Arg 180 185 190 Ile Asp Ile His Leu Arg Trp Ser Ser Arg LysGly Arg Lys Asp Arg 195 200 205 Glu Ile Arg Phe Thr Ala Lys 210 215 37746 DNA Brassica rapa 37 atgccgattg gtgaggttct tgtaggggct gctcttggaattacactcca agtgcttcat 60 gaagccatca taaaagcaaa acatagatct ttaaccacaaaatgtatctt ggaccgcctc 120 gatgctacaa tctccaggat cactccgttg gtggttcatgtcgataagat cagcaaaggg 180 gtagaagatt ctcagaggaa agtcattgaa gacctcaagcgtcttcttga aaaggctgtt 240 tttcttgttg aggcttatgc agaactcaga cgcagaaacctactcaagaa gtttaggtat 300 gtatagttta tatagtacat gaaatgcttg aaaagtctttgtgattctta aaatgttttt 360 gttttgttta tataatatat atgtgtgtgt tgttgatatctaggtacaag agtagaatca 420 aagagttgga agcttcttta agatggatgg tagaggtggatgttcaagtc aaccaatggt 480 tggatatcaa acaactcctg gccaagatgt ttgaaatgaacactaaactc gagaggatca 540 cgtgcccacc aactgattgt aattgtttca agagaaatgatagcacatca ccagtgatat 600 cacaaagtag taatcaaaat atactcgaag caacagacggatcgtcagag gaagacgaag 660 aagaaagccc aaggattgat atccaccttc gatggagttcaagaaaagga gctaaagatc 720 gtgagatccg attcatggtc aagtga 746 38 639 DNAArtificial Sequence Description of Artificial Sequence cDNA from B. rapa38 atgccgattg gtgaggttct tgtaggggct gctcttggaa ttacactcca agtgcttcat 60gaagccatca taaaagcaaa acatagatct ttaaccacaa aatgtatctt ggaccgcctc 120gatgctacaa tctccaggat cactccgttg gtggttcatg tcgataagat cagcaaaggg 180gtagaagatt ctcagaggaa agtcattgaa gacctcaagc gtcttcttga aaaggctgtt 240tttcttgttg aggcttatgc agaactcaga cgcagaaacc tactcaagaa gtttaggtac 300aagagtagaa tcaaagagtt ggaagcttct ttaagatgga tggtagaggt ggatgttcaa 360gtcaaccaat ggttggatat caaacaactc ctggccaaga tgtttgaaat gaacactaaa 420ctcgagagga tcacgtgccc accaactgat tgtaattgtt tcaagagaaa tgatagcaca 480tcaccagtga tatcacaaag tagtaatcaa aatatactcg aagcaacaga cggatcgtca 540gaggaagacg aagaagaaag cccaaggatt gatatccacc ttcgatggag ttcaagaaaa 600ggagctaaag atcgtgagat ccgattcatg gtcaagtga 639 39 212 PRT Brassica rapa39 Met Pro Ile Gly Glu Val Leu Val Gly Ala Ala Leu Gly Ile Thr Leu 1 510 15 Gln Val Leu His Glu Ala Ile Ile Lys Ala Lys His Arg Ser Leu Thr 2025 30 Thr Lys Cys Ile Leu Asp Arg Leu Asp Ala Thr Ile Ser Arg Ile Thr 3540 45 Pro Leu Val Val His Val Asp Lys Ile Ser Lys Gly Val Glu Asp Ser 5055 60 Gln Arg Lys Val Ile Glu Asp Leu Lys Arg Leu Leu Glu Lys Ala Val 6570 75 80 Phe Leu Val Glu Ala Tyr Ala Glu Leu Arg Arg Arg Asn Leu Leu Lys85 90 95 Lys Phe Arg Tyr Lys Ser Arg Ile Lys Glu Leu Glu Ala Ser Leu Arg100 105 110 Trp Met Val Glu Val Asp Val Gln Val Asn Gln Trp Leu Asp IleLys 115 120 125 Gln Leu Leu Ala Lys Met Phe Glu Met Asn Thr Lys Leu GluArg Ile 130 135 140 Thr Cys Pro Pro Thr Asp Cys Asn Cys Phe Lys Arg AsnAsp Ser Thr 145 150 155 160 Ser Pro Val Ile Ser Gln Ser Ser Asn Gln AsnIle Leu Glu Ala Thr 165 170 175 Asp Gly Ser Ser Glu Glu Asp Glu Glu GluSer Pro Arg Ile Asp Ile 180 185 190 His Leu Arg Trp Ser Ser Arg Lys GlyAla Lys Asp Arg Glu Ile Arg 195 200 205 Phe Met Val Lys 210 40 17 PRTArabidopsis thaliana 40 Met Ile Ala Glu Val Ala Ala Gly Gly Ala Leu GlyLeu Ala Leu Ser 1 5 10 15 Val 41 40 PRT Arabidopsis thaliana 41 Arg LeuLys Leu Leu Leu Glu Asn Ala Val Ser Leu Val Glu Glu Asn 1 5 10 15 AlaGlu Leu Arg Arg Arg Asn Val Arg Lys Lys Phe Arg Tyr Met Arg 20 25 30 AspIle Lys Glu Phe Glu Ala Lys 35 40 42 27 PRT Arabidopsis thaliana 42 ValAsp Val Gln Val Asn Gln Leu Ala Asp Ile Lys Glu Leu Lys Ala 1 5 10 15Lys Met Ser Glu Ile Ser Thr Lys Leu Asp Lys 20 25 43 215 PRT ArtificialSequence Description of Artificial Sequence Consensus 43 Met Pro Xaa XaaGlu Xaa Xaa Xaa Gly Ala Ala Leu Gly Leu Xaa Leu 1 5 10 15 Gln Xaa LeuHis Xaa Ala Xaa Xaa Xaa Ala Lys Asp Xaa Ser Xaa Xaa 20 25 30 Thr Xaa XaaIle Leu Xaa Arg Leu Xaa Ala Thr Ile Xaa Xaa Ile Thr 35 40 45 Pro Xaa XaaXaa Xaa Xaa Asp Lys Xaa Xaa Xaa Glu Xaa Xaa Xaa Ser 50 55 60 Xaa Xaa ArgLys Val Xaa Glu Xaa Leu Lys Xaa Leu Leu Glu Xaa Ala 65 70 75 80 Xaa XaaLeu Val Glu Ala Tyr Ala Glu Leu Xaa Arg Arg Asn Xaa Leu 85 90 95 Xaa LysXaa Arg Tyr Xaa Arg Xaa Ile Lys Glu Xaa Glu Xaa Xaa Leu 100 105 110 ArgTrp Xaa Xaa Asp Val Asp Val Gln Val Asn Gln Trp Xaa Asp Ile 115 120 125Lys Xaa Leu Xaa Ala Lys Met Ser Glu Met Xaa Thr Lys Leu Xaa Xaa 130 135140 Ile Xaa Xaa Gln Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 145150 155 160 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215 44 22 DNAArtificial Sequence Description of Artificial Sequence Primer 44gacccgtaca gtactaagtc ta 22 45 24 DNA Artificial Sequence Description ofArtificial Sequence Primer 45 gatttccgaa attgattaca agaa 24 46 24 DNAArtificial Sequence Description of Artificial Sequence Primer 46atgccgattg gtgagcttgc gata 24 47 24 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 47 tcaagctctt attttactac aagc 24 48 24 DNAArtificial Sequence Description of Artificial Sequence Primer 48aatggacact aaacttgctg aagt 24 49 19 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 49 ccacaactat tatgcttct 19 50 24 DNAArtificial Sequence Description of Artificial Sequence Primer 50gaaccaaaaa cggctcgata ctaa 24 51 33 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 51 ccggaattca tgccgattgg tgagcttgcg ata 3352 33 DNA Artificial Sequence Description of Artificial Sequence Primer52 cgcggatcct caagctctta ttttactaca agc 33 53 24 DNA Artificial SequenceDescription of Artificial Sequence Primer 53 aactcttcac ctcgagagct aaca24 54 24 DNA Artificial Sequence Description of Artificial SequencePrimer 54 agtcgtttga cacaattggg acat 24 55 21 DNA Artificial SequenceDescription of Artificial Sequence Primer 55 atgattgctg aggttgccgc a 2156 24 DNA Artificial Sequence Description of Artificial Sequence Primer56 tcaagaatca tcactgcaga acgt 24 57 24 DNA Artificial SequenceDescription of Artificial Sequence Primer 57 gctaaattac gatgggtggt agat24 58 24 DNA Artificial Sequence Description of Artificial SequencePrimer 58 cgatgggtgg tagatgtgga tgtt 24 59 16 DNA Artificial SequenceDescription of Artificial Sequence Primer 59 ggatcgcacg gtttgt 16 60 23DNA Artificial Sequence Description of Artificial Sequence Primer 60ctgaacttct tgcgtacgtt tct 23 61 30 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 61 ccggaattca tgattgctga ggttgccgca 30 6233 DNA Artificial Sequence Description of Artificial Sequence Primer 62ccgggatcct caagaatcat cactgcagaa cgt 33 63 11 PRT Arabidopsis thaliana63 Asp Ile Lys Glu Ile Lys Ala Lys Ile Ser Glu 1 5 10 64 24 DNAArtificial Sequence Description of Artificial Sequence Primer 64atccgcctct ttcttttggt tttc 24 65 23 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 65 gtgttacttt tctacagcca gag 23 66 22 DNAArtificial Sequence Description of Artificial Sequence Primer 66gtctgaatcc gtcaagcctt cg 22 67 23 DNA Artificial Sequence Description ofArtificial Sequence Primer 67 tccatgcttc tatattgaag agc 23 68 23 DNAArtificial Sequence Description of Artificial Sequence Primer 68gattgtatag gttggttgat gag 23 69 23 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 69 gcatctcatt gacctccctt atc 23 70 23 DNAArtificial Sequence Description of Artificial Sequence Primer 70cagcttcctt caccgtctca tgg 23 71 23 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 71 ccaggaaaat aacggtgacg atc 23 72 23 DNAArtificial Sequence Description of Artificial Sequence Primer 72gtcatcatct aaagaggata agg 23 73 23 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 73 ggttgaaaaa gtggctttgg atg 23 74 23 DNAArtificial Sequence Description of Artificial Sequence Primer 74atggatccgg cgactaattc acc 23 75 23 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 75 tgtcctcagg aatctcagag agc 23

1. An isolated nucleic acid molecule which nucleic acid consistsessentially of an RPW nucleotide sequence encoding an RPW resistancepolypeptide having an N-terminal transmembrane domain and a coiled coildomain and which is capable of recognising and activating in a plantinto which said nucleic acid is introduced a specific defense responseto challenge with a powdery mildew pathogen which is any of: E.cichoracearum, E. cruciferarum, E. orontii, Oidium lycopersici.
 2. Anucleic acid as claimed in claim 1 wherein the RPW nucleotide sequenceis derived from an RPW7 or RP8 locus in a plant.
 3. An isolated nucleicacid molecule which consists essentially of an RPW nucleotide sequencewhich: (i) encodes an RPW resistance polypeptide selected from any shownin Sequence listing 4 (RPW8.1) or Sequence listing 6 (RPW8.2) as Ms-0,Wa-1, Kas-1, or C24; or shown in Sequence listing 9 (BrHR1), 12 (BrHR2),or 15 (BrHR3), or in Example 4 (hr1, hr2, or hr3), or, (ii) encodes ahomologous variant of the RPW resistance polypeptide of (i), whichshares at least about 50%, 60%, 70%, 80% or 90% identity therewith, andwherein the nucleic acid encoding said homologous variant hybridises at37° C. in a formamide concentration of about 20% and a saltconcentration of about 5×SSC with any complement RPW nucleic acid havinga sequence selected from: RPW8.1 genomic sequence (shown as 13878 . . .14719 in Sequence Listing 2); RPW8.2 genomic sequence (shown as 15904 .. . 16829 in Sequence Listing 2); RPW8.1 cDNA sequence or RPW8.2 cDNAsequence complementary to that shown in Sequence listing 4 or Sequencelisting 6 as Ms-0, Wa-1, Kas-1, or C24; BrHR1 genomic or cDNA sequencecomplementary to that shown in Sequence listing 7 or 8, BrHR2 genomic orcDNA sequence complementary to that shown in Sequence listing 10 or 11,BrHR3 genomic or cDNA sequence complementary to that shown in Sequencelisting 13 or 14, HR1 genomic sequence (shown as 19087 . . . 20103 inSequence Listing 1); HR2 genomic sequence (shown as 20600 . . . 21408 inSequence Listing 1); HR3 genomic sequence (shown as 25912 . . . 26632 inSequence Listing 1).
 4. A nucleic acid as claimed in claim 3 wherein theRPW nucleotide sequence encodes an RPW resistance polypeptide selectedRPW8.1 or RPW8.2 sequences which are shown in Sequence Listing
 2. 5. Anucleic acid as claimed in any one of claims 1 to 3 wherein the RPWnucleotide sequence is selected from a list consisting of: RPW8.1genomic sequence (shown as 13878 . . . 14719 complement in SequenceListing 2); RPW8.2 genomic sequence (shown as 15904 . . . 16829complement in Sequence Listing 2); RPW8.1 cDNA sequence or RPW8.2 cDNAsequence shown in Sequence listing 4 or Sequence listing 6 as Ms-0,Wa-1, Kas-1, or C24; BrHR1 genomic or cDNA sequence shown in Sequencelisting 7 or 8, BrHR2 genomic or cDNA sequence shown in Sequence listing10 or 11, BrHR3 genomic or cDNA sequence shown in Sequence listing 13 or14, HR1 genomic sequence (shown as 19087 . . . 20103 complement inSequence Listing 1); HR2 genomic sequence (shown as 20600 . . . 21408complement in Sequence Listing 1); HR3 genomic sequence (shown as
 25912. . . 26632 complement in Sequence Listing 1).
 6. A nucleic acid asclaimed in claim 3 wherein the RPW nucleotide sequence encodes aderivative of an RPW resistance polypeptide of claim 3 (i) by way ofaddition, insertion, deletion or substitution of one or more aminoacids.
 7. A nucleic acid as claimed in claim 6 which wherein the encodedderivative comprises the sequence DIKEIKAKISE.
 8. A nucleic acid asclaimed in claim 3 wherein the RPW nucleotide sequence consists of anallelic, paralogous or orthologous variant of an RPW nucleotide sequenceof claim
 5. 9. A nucleic acid as claimed in claim 3 wherein the variantis obtainable from a plant selected from: barley; Brassica napus; B.oleracea.
 10. An isolated nucleic acid which consists essentially of anucleotide sequence which is the complement of the RPW nucleotidesequence of any one of the preceding claims.
 11. An isolated nucleicacid for use as a probe or primer, said nucleic acid consisting of adistinctive sequence of at least about 16-30 nucleotides in length,which sequence is (i) conserved between two or more cDNA nucleotidesequences of sequence listing 3 or sequence listing 5; (ii) a sequencedegeneratively equivalent to said conserved sequence, or (iii) thecomplement sequence of either.
 12. A nucleic acid primer as claimed inclaim 11 which encodes all or part of any one the following conservedamino acid motifs: DIKEIKAKISE; MIAEVAAGGA LGLALSV; RLKLLLENAVSLVEENAELR RRNVRKKFRY MRDIKEFEAK; VDVQ VNQLADIKEL KAKMSEISTK LDK.
 13. Anucleic acid primer for amplification of RPW8.1 selected from:GACCCGTACAGTACTAAGTCTA GATTTCCGAAATTGATTACAAGAA ATGCCGATTGGTGAGCTTGCGATATCAAGCTCTTATTTTACTACAAGC AATGGACACTAAACTTGCTGAAGT CCACAACTATTATGCTTCTGAACCAAAAACGGCTCGATACTAA CCGGAATTCATGCCGATTGGTGAGCTTGCGATACGCGGATCCTCAAGCTCTTATTTTACTACAAGC

or for amplification of RPW8.2 selected from: AACTCTTCACCTCGAGAGCTAACAAGTCGTTTGACACAATTGGGACAT ATGATTGCTGAGGTTGCCGCA TCAAGAATCATCACTGCAGAACGTGCTAAATTACGATGGGTGGTAGAT CGATGGGTGGTAGATGTGGATGTT GGATCGCACGGTTTGTCTGAACTTCTTGCGTACGTTTCT. CCGGAATTCATGATTGCTGAGGTTGCCGCACCGGGATCCTCAAGAATCATCACTGCAGAACGT


14. A method for identifying, cloning, or determining the presencewithin a plant of a nucleic acid as claimed in any one of claims 1 to 9,which method employs a nucleic acid as claimed in any one of claims 10to
 13. 15. A method as claimed in claim 14, which method comprises thesteps of: (a) providing a preparation of nucleic acid from a plant cell;(b) providing a nucleic acid molecule which is a nucleic acid as claimedin claim 10, (c) contacting nucleic acid in said preparation with saidnucleic acid molecule under conditions for hybridisation, and, (d)identifying nucleic acid in said preparation which hybridises with saidnucleic acid molecule.
 16. A method as claimed in claim 14, which methodcomprises the steps of: (a) providing a preparation of nucleic acid froma plant cell; (b) providing a pair of nucleic acid molecule primerssuitable for PCR, at least one of said primers being a primer of any oneof claims 11 to 13, (c) contacting nucleic acid in said preparation withsaid primers under conditions for performance of PCR, (d) performing PCRand determining the presence or absence, and optionally the sequence, ofan amplified PCR product.
 17. A recombinant vector which comprises thenucleic acid of any one of claims 1 to
 9. 18. A vector as claimed inclaim 17 wherein the nucleic acid is operably linked to a promoter fortranscription in a host cell, wherein the promoter is optionally aninducible promoter.
 19. A vector as claimed in claim 17 or claim 18which is a plant vector.
 20. A vector as claimed in claim 19 which isthe SE7.5 construct shown in FIG. 3 herein.
 21. A method which comprisesthe step of introducing the vector of any one of claims 17 to 20 into ahost cell, and optionally causing or allowing recombination between thevector and the host cell genome such as to transform the host cell. 22.A host cell containing or transformed with a heterologous vector of anyone of claims 17 to
 20. 23. A method for producing a transgenic plant,which method comprises the steps of: (a) performing a method as claimedin claim 22 wherein the host cell is a plant cell, (b) regenerating aplant from the transformed plant cell.
 24. A transgenic plant which isoptionally selected from a species which is susceptible to powderymildew, and which is obtainable by the method of claim 23, or which is aclone, or selfed or hybrid progeny or other descendant of saidtransgenic plant, which in each case includes a heterologous nucleicacid of any one of claims 1 to
 9. 25. A transgenic plant as claimed inclaim 24 which is selected from: wheat; barley; tomato; Nicotiana spp.26. A part of propagule from a plant as claimed in claim 24 or claim 25,and which in either case includes a heterologous nucleic acid of any oneof claims 1 to
 9. 27. An isolated polypeptide which is encoded by theRPW nucleotide sequence of any one of claims 1 to
 9. 28. A polypeptideas claimed in claim 27 which is an RPW resistance polypeptide selectedfrom any shown in Sequence listing 4 (RPW8.1) or Sequence listing 6(RPW8.2) as Ms-0, Wa-1, Kas-1, or C24; or shown in Sequence listing 9(BrHR1), 12 (BrHR2), or 15 (BrHR3), or in Example 4 (hr1, hr2, or hr3).29. A method of making the polypeptide of claim 27 or claim 26, whichmethod comprises the step of causing or allowing expression from anucleic acid of any one of claims 1 to 9 in a suitable host cell.
 30. Apolypeptide which comprises the antigen-binding site of an antibodyhaving specific binding affinity for the polypeptide of claim
 28. 31. Amethod for influencing or affecting the degree of resistance of a plantto a powdery mildew caused by any one of E. cichoracearurn,E.cruciferarum, E.orontii, Oidium lycopersici, which method comprisesthe step of causing or allowing expression of a heterologous nucleicacid as claimed in any one of claims 1 to 10 within the cells of theplant, following an earlier step of introducing the nucleic acid into acell of the plant or an ancestor thereof.
 32. A method as claimed inclaim 31 for increasing a plant's powdery mildew disease resistance,wherein the nucleic acid is a nucleic acid as claimed in any one ofclaims 1 to
 9. 33. An isolated nucleic acid molecule encoding thepromoter of an RPW nucleotide sequence of claim 5, or a homologousvariant thereof which has promoter activity which is operably linked toa heterologous coding sequence.
 34. A nucleic acid as claimed in claim33 wherein the promoter is wound and SA induced but not JA induced. 35.A nucleic acid as claimed in claim 33 wherein the promoter is that ofRPW8.1 (15904 to 14719) or RPW8.2 (16829 to 19087) of Sequence Listing1.